Product Citations: 130

Fig.1.D showing Western Blotting from the publication: An independent regulator of global release pathways in astrocytes generates a subtype of extracellular vesicles required for postsynaptic function.
Fig.1.C showing Western Blotting from the publication: MicroRNAs are minor constituents of extracellular vesicles that are rarely delivered to target cells.
Fig.1.C showing Western Blotting in a Homo sapiens (Human) sample from the publication: The Alkylating Agent Methyl Methanesulfonate Triggers Lipid Alterations at the Inner Nuclear Membrane That Are Independent from Its DNA-Damaging Ability.
Fig.7.B showing Western Blotting from the publication: Spatiotemporal processing of neural cell adhesion molecules 1 and 2 by BACE1 in vivo.
Fig.7.D showing Western Blotting from the publication: Spatiotemporal processing of neural cell adhesion molecules 1 and 2 by BACE1 in vivo.
Fig.7.A showing Western Blotting from the publication: Spatiotemporal processing of neural cell adhesion molecules 1 and 2 by BACE1 in vivo.
Fig.6.A showing Western Blotting from the publication: Spatiotemporal processing of neural cell adhesion molecules 1 and 2 by BACE1 in vivo.
Fig.5.A showing Western Blotting from the publication: Spatiotemporal processing of neural cell adhesion molecules 1 and 2 by BACE1 in vivo.
Fig.1.D showing Western Blotting from the publication: Spatiotemporal processing of neural cell adhesion molecules 1 and 2 by BACE1 in vivo.
Fig.5.E showing Western Blotting in a Mus musculus (House mouse) sample from the publication: BACE1 partial deletion induces synaptic plasticity deficit in adult mice.
Fig.4.B showing Western Blotting in a Homo sapiens (Human) sample from the publication: 24(S)-Hydroxycholesterol induces ER dysfunction-mediated unconventional cell death.
Fig.2.A showing Western Blotting in a Rattus norvegicus (Rat) sample from the publication: Connexin 43 plays an important role in the transformation of cholangiocytes with Clonochis sinensis excretory-secretory protein and N-nitrosodimethylamine.
Fig.3.A showing Western Blotting in a Rattus norvegicus (Rat) sample from the publication: Connexin 43 plays an important role in the transformation of cholangiocytes with Clonochis sinensis excretory-secretory protein and N-nitrosodimethylamine.
Fig.4.B showing Western Blotting in a Mus musculus (House mouse) sample from the publication: LPAAT3 incorporates docosahexaenoic acid into skeletal muscle cell membranes and is upregulated by PPARδ activation.
Fig.2.E showing Western Blotting in a Mus musculus (House mouse) sample from the publication: LPAAT3 incorporates docosahexaenoic acid into skeletal muscle cell membranes and is upregulated by PPARδ activation.
Fig.1.C showing Western Blotting in a Mus musculus (House mouse) sample from the publication: LPAAT3 incorporates docosahexaenoic acid into skeletal muscle cell membranes and is upregulated by PPARδ activation.
Fig.1.D showing Western Blotting in a Homo sapiens (Human) sample from the publication: Endosomal phosphatidylserine is critical for the YAP signalling pathway in proliferating cells.
Fig.3.C showing Western Blotting in a Mus musculus (House mouse) sample from the publication: Legionella effector Lpg1137 shuts down ER-mitochondria communication through cleavage of syntaxin 17.
Fig.3.B showing Western Blotting in a Mus musculus (House mouse) sample from the publication: Legionella effector Lpg1137 shuts down ER-mitochondria communication through cleavage of syntaxin 17.
Fig.3.A showing Western Blotting in a Mus musculus (House mouse) sample from the publication: Legionella effector Lpg1137 shuts down ER-mitochondria communication through cleavage of syntaxin 17.
Fig.6.B showing Western Blotting in a Mus musculus (House mouse) sample from the publication: Legionella effector Lpg1137 shuts down ER-mitochondria communication through cleavage of syntaxin 17.
Fig.1.C showing Western Blotting in a Mus musculus (House mouse) sample from the publication: Legionella effector Lpg1137 shuts down ER-mitochondria communication through cleavage of syntaxin 17.
Fig.5.C showing Western Blotting from the publication: The Polyherbal Wattana Formula Displays Anti-Amyloidogenic Properties by Increasing α-Secretase Activities.
Fig.1.A showing Western Blotting from the publication: Tetrahydrohyperforin Inhibits the Proteolytic Processing of Amyloid Precursor Protein and Enhances Its Degradation by Atg5-Dependent Autophagy.
Fig.10.G showing Western Blotting from the publication: Tagging strategies strongly affect the fate of overexpressed caveolin-1.
32 images found

Development of Broadly Applicable Reference EVs Expressing Horseradish Peroxidase.

In Journal of Extracellular Vesicles on 1 August 2025 by Waas, D., Juraschitz, M., et al.

Extracellular vesicles (EVs) hold great promise as circulating biomarkers for diagnostic and therapeutic approaches. Thus, many research groups world-wide investigate important aspects of EVs including their biology and medical significance. For this, a large number of procedures and protocols has been established making it difficult to almost impossible to compare and replicate results. Consequently, diagnostic tests remain problematic to interpret, mainly because the use of reliable reference EVs as a qualified standard has not yet gained widespread acceptance. Beyond doubt, such reference EVs are key to assess EV preparations quantitatively and to establish robust quality control processes to ensure overall quality and validity of data. To further advance the establishment of such controls, we designed and generated a new class of reference EVs expressing horseradish peroxidase (HRP) to facilitate simple and reliable EV tracing during isolation and standardization of EV purification and downstream analysis processes. HRP+ EVs can be quantified easily and with unmatched sensitivity either directly via measuring HRP activity or indirectly via immunodetection of HRP on the EV surface. We demonstrate that HRP+ EVs allow the reliable quantification of absolute EV numbers in biological or medical samples to normalize clinical specimens in liquid biopsies.
© 2025 Eximmium Biotechnologies GmbH. Journal of Extracellular Vesicles published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.

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S-palmitoylation modulates ATG2-dependent non-vesicular lipid transport during starvation-induced autophagy.

In The EMBO Journal on 1 May 2025 by Zheng, W., Pu, M., et al.

Lipid transfer proteins mediate the non-vesicular transport of lipids at membrane contact sites to regulate the lipid composition of organelle membranes. Despite significant recent advances in our understanding of the structural basis for lipid transfer, its functional regulation remains unclear. In this study, we report that S-palmitoylation modulates the cellular function of ATG2, a rod-like lipid transfer protein responsible for transporting phospholipids from the endoplasmic reticulum (ER) to phagophores during autophagosome formation. During starvation-induced autophagy, ATG2A undergoes depalmitoylation as the balance between ZDHHC11-mediated palmitoylation and APT1-mediated depalmitoylation. Inhibition of ATG2A depalmitoylation leads to impaired autophagosome formation and disrupted autophagic flux. Further, in cell and in vitro analyses demonstrate that S-palmitoylation at the C-terminus of ATG2A anchors the C-terminus to the ER. Depalmitoylation detaches the C-terminus from the ER membrane, enabling it to interact with phagophores and promoting their growth. These findings elucidate a S-palmitoylation-dependent regulatory mechanism of cellular ATG2, which may represent a broad regulatory strategy for lipid transport mediated by bridge-like transporters within cells.
© 2025. The Author(s).

  • Cell Biology
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Continuous collection of human mesenchymal-stromal-cell-derived extracellular vesicles from a stirred tank reactor operated under xenogeneic-free conditions for therapeutic applications.

In Stem Cell Research & Therapy on 24 April 2025 by Ulpiano, C., Salvador, W., et al.

Mesenchymal-stromal-cell-derived extracellular vesicles (MSC-EVs) play a key role in the paracrine effects of MSC and have demonstrated therapeutic potential in various preclinical models. However, clinical translation is hindered by manufacturing practices relying on planar culture systems, fetal bovine serum (FBS)-supplemented media, and non-scalable, low-purity EV isolation methods that fail to meet dose and safety requirements, underscoring the need for innovative approaches. In this study, we developed a scalable platform to manufacture human MSC-EVs at clinically relevant numbers, integrating continuous collection of EV-enriched conditioned media (CM) using a stirred-tank reactor (STR) under xenogeneic-free conditions and a scalable downstream process.
Wharton's jelly-derived MSC (MSC(WJ)) were expanded using microcarriers in a controlled STR using human platelet lysate (hPL)-supplemented medium. Then, a 3-day EV production stage, featuring continuous harvesting of the CM, was established using a novel serum-/xeno(geneic)-free exosome depleted-hPL supplement. For the isolation of MSC-EVs, a scalable process was implemented by pairing tangential flow filtration and anion exchange chromatography. Isolated MSC-EVs were characterised using nanoparticle tracking analysis, protein and zeta potential quantification, western blot analysis of EV protein markers, transmission electron microscopy and uptake studies of fluorescently labelled-EVs.
The system sustained the efficient expansion of MSC(WJ), reaching a total of (6.03 ± 0.181) x 107 cells after 7 days, which corresponds to a 30.1 ± 0.740-fold expansion. Upon a 3-day continuous CM harvesting, a total of (2.13 ± 0.301) x 1012 EVs were isolated corresponding to a particle yield factor of (1.26 ± 0.186) x 104 EVs/cell/day. MSC-EVs presented high purity levels ((5.53 ± 1.55) x 109 particles/µg), a homogeneous small size distribution (mean diameter of 115 ± 4.88 nm), a surface charge of -23.4 ± 6.23 mV, positive detection of tetraspanins CD9 and CD63 and syntenin-1 and displayed a typical cup-shaped morphology. MSC-EVs were readily incorporated by endothelial cells and two human breast cancer cell lines.
Overall, the scalable and Good Manufacturing Practices (GMP)-compliant platform established herein enabled the reproducible manufacturing of MSC-EVs with high purity and generally accepted characteristics concerning size, protein markers, surface charge, morphology, and cellular internalization, validating its potential for future clinical applications.
© 2025. The Author(s).

  • WB
  • Homo sapiens (Human)
  • Stem Cells and Developmental Biology
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Global functional genomics reveals GRK5 as a cystic fibrosis therapeutic target synergistic with current modulators.

In IScience on 21 March 2025 by Botelho, H. M., Lopes-Pacheco, M., et al.

Cystic fibrosis (CF) is a life-shortening disease affecting >160,000 individuals worldwide predominantly with respiratory symptoms. About 80% of individuals with CF have the p.Phe508del variant that causes the CF transmembrane conductance regulator (CFTR) protein to misfold and be targeted for premature degradation by the endoplasmic reticulum (ER) quality control (ERQC), thus preventing its plasma membrane (PM) traffic. Despite the recent approval of a "highly effective" drug rescuing p.Phe508del-CFTR, maximal lung function improvement is ∼14%. To identify global modulators of p.Phe508del traffic, we performed a high-content small interfering RNA (siRNA) microscopy-based screen of >9,000 genes and monitored p.Phe508del-CFTR PM rescue in human airway cells. This primary screen identified 227 p.Phe508del-CFTR traffic regulators, of which 35 could be validated by additional siRNAs. Subsequent mechanistic studies established GRK5 as a robust regulator whose inhibition rescues p.Phe508del-CFTR PM traffic and function in primary and immortalized cells, thus emerging as a novel potential drug target for CF.
© 2025 The Author(s).

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SARS-CoV-2 predation of Golgi-bound PI4P primes the massive activation of the DNA Damage Response kinase ATM in the cytoplasm

Preprint on BioRxiv : the Preprint Server for Biology on 6 December 2024 by Rebendenne, A., Soulet, C., et al.

Like all viruses, SARS-CoV-2, the causative agent of COVID-19, relies on host cell resources to replicate. Our study reveals that, among these resources, SARS-CoV-2 hijacks the oxysterol-binding protein 1 (OSBP1) transporter to exploit the Golgi-bound phosphatidylinositol-4-phosphate (PI4P) pool. This leads to a depletion of Golgi-resident PI4P, triggering the activation of the ATM DNA Damage Response (DDR) kinase in the cytoplasm. As such, ATM, typically anchored to PI4P at the Golgi in an inactive state, undergoes auto-phosphorylation and cytoplasmic release upon SARS-CoV-2-induced PI4P depletion. Conversely, pharmacological inhibition of ATM auto-phosphorylation, which stabilizes its interaction with PI4P, significantly impairs SARS-CoV-2 replication. The requirement for PI4P and impact of ATM inhibition might be conserved across coronaviruses, as similar effects were observed with HCoV-229E. Finally, SARS-CoV-2-induced, cytoplasmic ATM pre-activation primes cells for an accelerated response to DNA damage, which might contribute to the severe outcomes of COVID-19 observed in cancer patients undergoing chemo- or radiotherapy. Therefore, this study uncovers a DNA damage-independent mode of ATM activation and highlights the potential of ATM inhibitors as therapeutic agents against COVID-19.

  • Cell Biology
  • COVID-19
  • Genetics
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An independent regulator of global release pathways in astrocytes generates a subtype of extracellular vesicles required for postsynaptic function.

In Sci Adv on 23 June 2023 by Levy-Myers, R., Daudelin, D., et al.

Fig.1.D
Fig.1.D showing Western Blotting from the publication: An independent regulator of global release pathways in astrocytes generates a subtype of extracellular vesicles required for postsynaptic function.
  • WB
GDE3 induces the release of MVs in HEK293T cells.(A) Graph quantifying normalized EV concentration from transfected HEK293T cells. (B) Representative image of HEK293T cells stably expressing GDE3 or transfected GDE3-ΔN. FLAG (GDE3, green) and DAPI (blue). Scale bar, 20 μm. (C) Representative elec... more
GDE3 induces the release of MVs in HEK293T cells.(A) Graph quantifying normalized EV concentration from transfected HEK293T cells. (B) Representative image of HEK293T cells stably expressing GDE3 or transfected GDE3-ΔN. FLAG (GDE3, green) and DAPI (blue). Scale bar, 20 μm. (C) Representative electron microscope (EM) images of 12,000 and 100,000 EVs from HEK293T cells transfected with vector or plasmids expressing GDE3. Scale bar: 100 nm. Histogram of sizes of 12,000 and 100,000 EVs from vector-transfected HEK293T cells visualized by EM. (D) Representative Western blots and graphs (E to H) quantifying 12,000 and 100,000 EVs from HEK293T cells transfected with the indicated plasmids. Long and short refer to exposure times. (E) MV marker annexin A1 was quantified in 12,000 EVs; exosome markers CD63 (F) and CD81 (G) were quantified in 100,000 EVs [see fig. S1 (C to E) for additional quantification]; (H) GDE3 (FLAG) was quantified in 12,000 and 100,000 EVs. All graphs are means ± SEM. Data points are biological replicates. ns, P > 0.05; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. See table S1 for statistical details. ns, not significant. less

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MicroRNAs are minor constituents of extracellular vesicles that are rarely delivered to target cells.

In PLoS Genet on 1 December 2021 by Albanese, M., Chen, Y. A., et al.

Fig.1.C
Fig.1.C showing Western Blotting from the publication: MicroRNAs are minor constituents of extracellular vesicles that are rarely delivered to target cells.
  • WB
Isolation and characterization of extracellular vesicles from EBV-infected cells.(A) Schematic overview of our method of extracellular vesicles (EVs) enrichment and purification. EBV-infected B cells, lymphoblastoid cell lines (LCLs), were seeded at an initial density of 0.5×106 cells/ml in cell-... more
Isolation and characterization of extracellular vesicles from EBV-infected cells.(A) Schematic overview of our method of extracellular vesicles (EVs) enrichment and purification. EBV-infected B cells, lymphoblastoid cell lines (LCLs), were seeded at an initial density of 0.5×106 cells/ml in cell-culture medium containing 2% of EV-depleted fetal calf serum (see Materials and Methods) and processed as indicated. (B) The concentrations of EVs in the 10 fractions after iodixanol (Optiprep) floating density gradient centrifugation were measured by nanoparticle tracking analysis (NTA). One representative preparation is shown. (C) Western blot immunodetection of the EV marker protein TSG101 and the EBV protein LMP1, which are enriched in EVs, and Calnexin as a negative control in the 10 fractions. 5 μg of the cell lysate (CL) or resuspended ‘miniUC pellet’ (mini UC) preparations as indicated in panel A were used as controls. Per density gradient fraction 10, 20, or 60 μl were loaded onto the gels to detect LMP1, TSG101, or Calnexin, respectively. One representative preparation of three is shown. (D) Relative levels of four selected miRNAs (three viral miRNAs and a representative human miRNA) were analyzed by TaqMan RT-qPCR analysis to determine their physical density characteristics after floating density ultracentrifugation. All miRNAs are found in fractions 2–4. One representative quantification of three is shown. (E) Electron microscopic analysis of negative-stained EVs after iodixanol density gradient purification. Scale bars are 200 nm. less

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The Alkylating Agent Methyl Methanesulfonate Triggers Lipid Alterations at the Inner Nuclear Membrane That Are Independent from Its DNA-Damaging Ability.

In Int J Mol Sci on 12 July 2021 by Ovejero, S., Soulet, C., et al.

Fig.1.C
Fig.1.C showing Western Blotting in a Homo sapiens (Human) sample from the publication: The Alkylating Agent Methyl Methanesulfonate Triggers Lipid Alterations at the Inner Nuclear Membrane That Are Independent from Its DNA-Damaging Ability.
  • WB
  • Homo sapiens (Human)
MMS triggers the formation of nuclear LD in RPE-1 cells. (A) Microscopy images corresponding to RPE-1 cells treated for 2 h with 0.005% MMS. Nuclei are identified by staining DNA with DAPI, and LD with the vital dye NileRed. To facilitate visualization of the respective position of the LD within ... more
MMS triggers the formation of nuclear LD in RPE-1 cells. (A) Microscopy images corresponding to RPE-1 cells treated for 2 h with 0.005% MMS. Nuclei are identified by staining DNA with DAPI, and LD with the vital dye NileRed. To facilitate visualization of the respective position of the LD within the nucleus, merged images are shown at the bottom, and the perimeter of nuclei superimposed as dashed lines onto the LD images. (B) Quantification of the number of nuclear LD counted in RPE-1 cells upon treatment with the indicated agents. When cerulenin is indicated, it was added at 5 μg/mL to the cultures for 4 h, plus the incubation or not with 0.005% MMS, 100 μg/mL zeocin or 1 mM HU during the last 2 h. Each dot corresponds to the value noted for one nucleus. At least 200 nuclei per experiment and condition were counted from 3 independent experiments. The boxes comprise the 10–90 percentile values, and their central bar represents the median of the population. Since populations were not normally distributed, the significance of differences between the populations is indicated by asterisks (*) and their associated p-values, obtained after applying a Mann–Whitney nonparametric test. (C) Western blot of extracts from RPE-1 cells untreated or treated with either 0.005% MMS, 100 μg/mL zeocin or 1 mM HU for the indicated times to monitor the activation of the DDR by following the phosphorylation on Serine 345 (pSer345) of the effector Chk1 and that of Chk2 on threonine 68 (pThr68). Molecular weights are shown in kDa as indicated by co-run molecular weight ladders. Red arrows mark the samples chosen for the microscopy studies shown in (B). (D) Details as in (A) for cells treated 4 h with cerulenin at 5 μg/mL, and then 0.005% MMS added during the last 2 h. Dashed white lines define the nucleus boundaries. Yellow arrowheads point at the locations of the nuclei periphery from which LD are seen to access the nucleus, eventually preventing the DAPI staining. less

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Spatiotemporal processing of neural cell adhesion molecules 1 and 2 by BACE1 in vivo.

In J Biol Chem on 7 February 2021 by Kim, W., Watanabe, H., et al.

Fig.7.B
Fig.7.B showing Western Blotting from the publication: Spatiotemporal processing of neural cell adhesion molecules 1 and 2 by BACE1 in vivo.
  • WB
PSA-NCAM1 levels are increased in BACE1−/− mice at P10. PBS soluble (Soluble) and membrane (Membrane) fractions of hippocampus (HC) and olfactory bulb (OB) samples from postnatal day 10 (P10), 4-month-old (4 months), and 12-month-old (12 months) BACE1+/+ and BACE1−/− mice were analyzed by immunob... more
PSA-NCAM1 levels are increased in BACE1−/− mice at P10. PBS soluble (Soluble) and membrane (Membrane) fractions of hippocampus (HC) and olfactory bulb (OB) samples from postnatal day 10 (P10), 4-month-old (4 months), and 12-month-old (12 months) BACE1+/+ and BACE1−/− mice were analyzed by immunoblot with anti-PSA-NCAM1 (2-2B), anti-C-terminal NCAM1 (AB5032), anti-N-terminal NCAM1 (AF2408), anti-BACE1 (D10E5), anti-calnexin (610523), and GAPDH (MAB374) antibodies. A, PSA-NCAM1 levels were analyzed in HC and OB of BACE1+/+ and BACE1−/− mice during aging. PSA-NCAM1 is predominantly present at P10, especially in the HC. Although PSA modification was significantly decreased with age, total NCAM1 levels remained unchanged. B, PSA-NCAM1 levels in HC and OB from BACE1−/− mice were higher than ones in BACE1+/+ mice at P10. Similarly, soluble PSA-NCAM1 (PSA-sNCAM1) levels in the HC from BACE1−/− mice were higher than ones in BACE1+/+ mice at P10. C, graphs represent densitometry of PSA-NCAM1 normalized to calnexin and PSA-sNCAM1 normalized to GAPDH. D, after treatment of sialidase to remove the sialic acid from NCAM1, total NCAM1 levels were similar between BACE1+/+ and BACE1−/− mice at P10. Unpaired two tailed t-test was used for analysis. ∗p < 0.05, ∗∗p < 0.001, (BACE1+/+; n = 5, BACE1−/−; n = 5). less

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Spatiotemporal processing of neural cell adhesion molecules 1 and 2 by BACE1 in vivo.

In J Biol Chem on 7 February 2021 by Kim, W., Watanabe, H., et al.

Fig.7.D
Fig.7.D showing Western Blotting from the publication: Spatiotemporal processing of neural cell adhesion molecules 1 and 2 by BACE1 in vivo.
  • WB
PSA-NCAM1 levels are increased in BACE1−/− mice at P10. PBS soluble (Soluble) and membrane (Membrane) fractions of hippocampus (HC) and olfactory bulb (OB) samples from postnatal day 10 (P10), 4-month-old (4 months), and 12-month-old (12 months) BACE1+/+ and BACE1−/− mice were analyzed by immunob... more
PSA-NCAM1 levels are increased in BACE1−/− mice at P10. PBS soluble (Soluble) and membrane (Membrane) fractions of hippocampus (HC) and olfactory bulb (OB) samples from postnatal day 10 (P10), 4-month-old (4 months), and 12-month-old (12 months) BACE1+/+ and BACE1−/− mice were analyzed by immunoblot with anti-PSA-NCAM1 (2-2B), anti-C-terminal NCAM1 (AB5032), anti-N-terminal NCAM1 (AF2408), anti-BACE1 (D10E5), anti-calnexin (610523), and GAPDH (MAB374) antibodies. A, PSA-NCAM1 levels were analyzed in HC and OB of BACE1+/+ and BACE1−/− mice during aging. PSA-NCAM1 is predominantly present at P10, especially in the HC. Although PSA modification was significantly decreased with age, total NCAM1 levels remained unchanged. B, PSA-NCAM1 levels in HC and OB from BACE1−/− mice were higher than ones in BACE1+/+ mice at P10. Similarly, soluble PSA-NCAM1 (PSA-sNCAM1) levels in the HC from BACE1−/− mice were higher than ones in BACE1+/+ mice at P10. C, graphs represent densitometry of PSA-NCAM1 normalized to calnexin and PSA-sNCAM1 normalized to GAPDH. D, after treatment of sialidase to remove the sialic acid from NCAM1, total NCAM1 levels were similar between BACE1+/+ and BACE1−/− mice at P10. Unpaired two tailed t-test was used for analysis. ∗p < 0.05, ∗∗p < 0.001, (BACE1+/+; n = 5, BACE1−/−; n = 5). less

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Spatiotemporal processing of neural cell adhesion molecules 1 and 2 by BACE1 in vivo.

In J Biol Chem on 7 February 2021 by Kim, W., Watanabe, H., et al.

Fig.7.A
Fig.7.A showing Western Blotting from the publication: Spatiotemporal processing of neural cell adhesion molecules 1 and 2 by BACE1 in vivo.
  • WB
PSA-NCAM1 levels are increased in BACE1−/− mice at P10. PBS soluble (Soluble) and membrane (Membrane) fractions of hippocampus (HC) and olfactory bulb (OB) samples from postnatal day 10 (P10), 4-month-old (4 months), and 12-month-old (12 months) BACE1+/+ and BACE1−/− mice were analyzed by immunob... more
PSA-NCAM1 levels are increased in BACE1−/− mice at P10. PBS soluble (Soluble) and membrane (Membrane) fractions of hippocampus (HC) and olfactory bulb (OB) samples from postnatal day 10 (P10), 4-month-old (4 months), and 12-month-old (12 months) BACE1+/+ and BACE1−/− mice were analyzed by immunoblot with anti-PSA-NCAM1 (2-2B), anti-C-terminal NCAM1 (AB5032), anti-N-terminal NCAM1 (AF2408), anti-BACE1 (D10E5), anti-calnexin (610523), and GAPDH (MAB374) antibodies. A, PSA-NCAM1 levels were analyzed in HC and OB of BACE1+/+ and BACE1−/− mice during aging. PSA-NCAM1 is predominantly present at P10, especially in the HC. Although PSA modification was significantly decreased with age, total NCAM1 levels remained unchanged. B, PSA-NCAM1 levels in HC and OB from BACE1−/− mice were higher than ones in BACE1+/+ mice at P10. Similarly, soluble PSA-NCAM1 (PSA-sNCAM1) levels in the HC from BACE1−/− mice were higher than ones in BACE1+/+ mice at P10. C, graphs represent densitometry of PSA-NCAM1 normalized to calnexin and PSA-sNCAM1 normalized to GAPDH. D, after treatment of sialidase to remove the sialic acid from NCAM1, total NCAM1 levels were similar between BACE1+/+ and BACE1−/− mice at P10. Unpaired two tailed t-test was used for analysis. ∗p < 0.05, ∗∗p < 0.001, (BACE1+/+; n = 5, BACE1−/−; n = 5). less

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Spatiotemporal processing of neural cell adhesion molecules 1 and 2 by BACE1 in vivo.

In J Biol Chem on 7 February 2021 by Kim, W., Watanabe, H., et al.

Fig.6.A
Fig.6.A showing Western Blotting from the publication: Spatiotemporal processing of neural cell adhesion molecules 1 and 2 by BACE1 in vivo.
  • WB
BACE1 proteolysis of SEZ6, APP, and CHL1 in the hippocampus and olfactory bulb with age.A, representative immunoblot of PBS soluble (Soluble) and membrane (Membrane) fractions of the hippocampus (HC) and olfactory bulb (OB) samples from postnatal day 10 (P10), 4-month-old (4 months), and 12-month... more
BACE1 proteolysis of SEZ6, APP, and CHL1 in the hippocampus and olfactory bulb with age.A, representative immunoblot of PBS soluble (Soluble) and membrane (Membrane) fractions of the hippocampus (HC) and olfactory bulb (OB) samples from postnatal day 10 (P10), 4-month-old (4 months), and 12-month-old (12 months) BACE1+/+ and BACE1−/− mice. Well-characterized BACE1 substrates are also analyzed in the hippocampus and olfactory bulb at three different ages using anti-SEZ6 (14E5; kindly provided by Dr S. Lichtenthaler), anti-CHL1 (AF2147), anti-sAPPβ (BAWT; kindly provided by Dr S. Lichtenthaler), anti-APP (C1/6.1), anti-NCAM1 (AF-2408), anti-NCAM2 (sc-136328), anti-BACE1 (D10E5), anti-GAPDH (MAB374), and anti-calnexin (610523) antibodies. The molecular weight of sSEZ6β in HC (∼155 kDa, arrowhead) is different from the one in the OB (∼165 kDa, double arrowheads) at the age of 4 and 12 months. The weak band at ∼140 kDa (asterisk) represents immature SEZ6-FL, while the strong band at 165-kDa (open arrowhead) or 175-kDa (double open arrowheads) in the OB represents mature SEZ6-FL. The different molecular weight of sSEZ6β or SEZ6-FL between the HC and OB of 4- and 12-month-old mice is most likely due to alternative splicing or posttranslational modifications. In the HC membrane fraction, CHL1-FL was detected at ∼185 kDa at P10, while CHL1-FL is detected as two bands at ∼185 kDa (plain arrow) and ∼175 kDa (dash arrow) in 4- and 12-month-old mice, most likely due to splicing variant or glycosylation. B–G, graphs represent densitometry of soluble protein (sSEZ6β, sAPPβ, and sCHL1) normalized to GAPDH or densitometry of membrane protein (mature SEZ6-FL, APP-FL, and 185-kDa CHL1) normalized to calnexin. White and gray bars represent BACE1+/+ and BACE1−/− mice, respectively. Unpaired two tailed t-test was used for analysis. ∗p < 0.05, ∗∗<0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, ns; not significant, P10 (BACE1+/+; n = 4–5, BACE1−/−; n = 4–5), 4 months (BACE1+/+; n = 5–6, BACE1−/−; n = 5), 12 months (BACE1+/+; n = 4–5, BACE1−/−; n = 4–5). less

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Spatiotemporal processing of neural cell adhesion molecules 1 and 2 by BACE1 in vivo.

In J Biol Chem on 7 February 2021 by Kim, W., Watanabe, H., et al.

Fig.5.A
Fig.5.A showing Western Blotting from the publication: Spatiotemporal processing of neural cell adhesion molecules 1 and 2 by BACE1 in vivo.
  • WB
Differential spatiotemporal BACE1 processing of NCAM1 and NCAM2 in vivo.A, representative immunoblot of PBS soluble (Soluble) and membrane (Membrane) fraction of the hippocampus (HC) and olfactory bulb (OB) samples from postnatal day 10 (P10), 4-month-old (4 months), and 12-month-old (12 months) ... more
Differential spatiotemporal BACE1 processing of NCAM1 and NCAM2 in vivo.A, representative immunoblot of PBS soluble (Soluble) and membrane (Membrane) fraction of the hippocampus (HC) and olfactory bulb (OB) samples from postnatal day 10 (P10), 4-month-old (4 months), and 12-month-old (12 months) BACE1+/+ and BACE1−/− mice. Well-characterized BACE1 substrates in vivo are also analyzed in the HC and OB at three different ages using anti-NCAM1 (AF-2408), anti-NCAM2 (sc-136328), BACE1 (D10E5), anti-calnexin (610523), and anti-GAPDH (MAB374) antibodies. After increasing the contrast of the image for sNCAM1, sNCAM1β was detected in HC and OB of BACE1+/+ mice at P10. Transmembrane (TM) and GPI-anchored (GPI) NCAM2 isoforms were observed in the OB membrane fraction, while only NCAM2-TM was detected in HC membrane fraction. Full-length NCAM1 levels (180, 140, and 120) were observed in membrane fraction. Notably, full-length NCAM1 levels are significantly decreased at P10. B–I, graphs represent densitometry of soluble protein normalized to GAPDH or densitometry of membrane protein normalized to calnexin. White and gray bars represent BACE1+/+ and BACE1−/− mice, respectively. Unpaired two tailed t-test was used for analysis. ∗p < 0.05, ∗∗<0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, ns, not significant, N.A.; not applicable, P10 (BACE1+/+; n = 4–5, BACE1−/−; n = 4–5), 4 months (BACE1+/+; n = 5–6, BACE1−/−; n = 5), 12 months (BACE1+/+; n = 4–5, BACE1−/−; n = 4–5). Full-length versions of the western blots (sNCAM2β and sNCAM1β) of 4-months-old mice shown in Figure S1, A and C. less

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Spatiotemporal processing of neural cell adhesion molecules 1 and 2 by BACE1 in vivo.

In J Biol Chem on 7 February 2021 by Kim, W., Watanabe, H., et al.

Fig.1.D
Fig.1.D showing Western Blotting from the publication: Spatiotemporal processing of neural cell adhesion molecules 1 and 2 by BACE1 in vivo.
  • WB
NCAM2 and NCAM1 are BACE1 substrates in the mouse olfactory bulb.A–B, schematic diagram of the two mouse NCAM2 (transmembrane [TM] and GPI-anchored) isoforms (A) and three major mouse NCAM1 (transmembrane [NCAM1-180 and NCAM1-140] and GPI-anchored [NCAM1-120]) isoforms (B) resulting from alternat... more
NCAM2 and NCAM1 are BACE1 substrates in the mouse olfactory bulb.A–B, schematic diagram of the two mouse NCAM2 (transmembrane [TM] and GPI-anchored) isoforms (A) and three major mouse NCAM1 (transmembrane [NCAM1-180 and NCAM1-140] and GPI-anchored [NCAM1-120]) isoforms (B) resulting from alternative splicing. NCAM2 and NCAM1 have five immunoglobulin (Ig)-like domains represented by ovals and two fibronectin type III repeats (FN) represented by rectangles. C, schematic presentation of NCAM2-TM and polysialylated NCAM1-140 illustrating antibody-binding sites. NCAM2 contains eight putative Asn-linked glycosylation sites (N-glycosylation) indicated by blue triangles, while NCAM1 has six N-glycosylation sites. In the fifth Ig (Ig5) of NCAM1, two of three N-glycosylation sites are polysialylated, represented by a chain of green circles (sialic acid). D, representative immunoblot of PBS soluble fraction (Soluble) and membrane fraction (Membrane) of olfactory bulb samples from 4-month-old BACE1+/+ and BACE1−/− mice using anti-NCAM2 (sc-136328), anti-NCAM1 (AF-2408), BACE1 (D10E5), GAPDH (MAB374), and anti-calnexin (610523) antibodies. BACE1-specific soluble NCAM2 (sNCAM2β) and NCAM1 (sNCAM1β) are observed in BACE1+/+ mice, but not in BACE1−/− mice. Full-length NCAM1 (NCAM1-180 and NCAM1-140, but not NCAM1-120) levels were slightly increased in BACE1−/−, compared with BACE1+/+ mice. However, levels of full-length NCAM2 (NCAM2-TM and NCAM2-GPI) were similar in BACE1+/+ and BACE1−/− mice. E–G, representative immunoblot of membrane fractions of olfactory bulb samples from 4-month-old BACE1+/+ and BACE1−/− mice using two different anti-C-terminal NCAM1 (rabbit pAb; AB5032 [E] and mouse mAb; 0B11 [F]) and anti-C-terminal NCAM2 (goat pAb; GTX89311 [G]) antibodies. After longer exposure, a ∼75 kDa NCAM1-CTF was detected in BACE1+/+ mice, but it was greatly decreased in BACE1−/− mice, and thus termed NCAM1-βCTF (arrow in D and E). However, a BACE1-specific NCAM2-βCTF was not observed (G). H, sagittal section from 12-month-old BACE1+/+ olfactory bulb was stained with anti-BACE1 (3D5, magenta), anti-NCAM2 (AF778, green), and anti-NCAM1 (AB5032, red) antibodies. NCAM1 and NCAM2 immunostaining with BACE1 are significantly overlapped in glomeruli. Scale bar represents 100 μm. D (BACE1+/+; n = 5–6, BACE1−/−; n = 5); E–G (BACE1+/+; n = 3 and BACE1−/−; n = 3); H (BACE1+/+; n = 4, two sagittal (H) and two coronal [Fig. S3] sections). less

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BACE1 partial deletion induces synaptic plasticity deficit in adult mice.

In Sci Rep on 27 December 2019 by Lombardo, S., Chiacchiaretta, M., et al.

Fig.5.E
Fig.5.E showing Western Blotting in a Mus musculus (House mouse) sample from the publication: BACE1 partial deletion induces synaptic plasticity deficit in adult mice.
  • WB
  • Mus musculus (House mouse)
Axon guidance defects were absent in hippocampus mossy fibers of aged BACE1 cKO mice following partial BACE1 deletion. (a) Coronal sections collected from aged mice were stained with anti-synaptoporin (SPO) antibody (green) and DAPI (blue). Scale bar 50 μm. (b) Quantification of IPB length showed... more
Axon guidance defects were absent in hippocampus mossy fibers of aged BACE1 cKO mice following partial BACE1 deletion. (a) Coronal sections collected from aged mice were stained with anti-synaptoporin (SPO) antibody (green) and DAPI (blue). Scale bar 50 μm. (b) Quantification of IPB length showed no alteration in TAM-treated mice compared to controls. IPB length was normalized on the length of the CA3 stratum lucidum (VEH n = 8; TAM n = 7, 3 to 4 sections per mouse). (c) Representative microscopy images showing reduced BACE1 (D10E5) expression in the hippocampus of TAM-treated mice. BACE1 signal was totally absent in BACE−/− mice, used as control to evaluate the amount of background in the staining. Scale bar 200 μm. Hippocampus full homogenates from TAM- or VEH-treated mice were resolved by SDS-PAGE for analysis of APP processing and fractionated (soluble and membrane fractions) for the analysis of SEZ6 and CHL1 processing. Homogenates from aged-matched BACE+/− and BACE1−/− were loaded as control samples. Representative blots of (d) APP-FL (C1/6.1) and APP- CTFs (C1/6.1), (e) fractionation blots of sAPPβ (BAWT), SEZ6 (14E5) and CHL1 (AF2147). (f) Densitometry analysis of protein expression. APP processing was reduced in TAM-treated mice as demonstrated by the accumulation of APP-FL (C1/6.1), and reduced levels of the βCTFs pC99 and pC89, and sAPPβ. βCTFs and sAPPβ were identified because missing in the BACE1−/− sample. SEZ6 processing was decreased in TAM-treated mice with accumulation of the full length and decreased levels of the ectodomain (SEZ6-NTF) as well as decreased SEZ6-NTF/SEZ6FL ratio. Processing of CHL1 was also impaired as showed by increased of CHL1-FL levels, while CHL1-NTF was not altered. CHL1-NTF/CHL1-FL ratio was significantly decreased. APP-FL, CTFs, SEZ6-NTF and CHL1-NTF were normalized to GAPDH (MAB374), SEZ6-FL and CHL1-FL were normalized to Calnexin (610523) (VEH n = 5; TAM n = 5). (g) Aβx-40 was quantified from hippocampus homogenates by MSD immunoassay. TAM-treated group displayed a significant reduction of Aβx-40 levels (~50% decrease) compared to control (VEH n = 7; TAM n = 7). Results were plotted as Mean ± SEM, **p < 0.005; ***p < 0.001; n.s. = not significant, Student’s t test. DG: dentate gyrus, IPB: infrapyramidal bundle, slu: stratum lucidum, MB: main bundle. less

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24(S)-Hydroxycholesterol induces ER dysfunction-mediated unconventional cell death.

In Cell Death Discov on 10 July 2019 by Urano, Y., Vo, D. H., et al.

Fig.4.B
Fig.4.B showing Western Blotting in a Homo sapiens (Human) sample from the publication: 24(S)-Hydroxycholesterol induces ER dysfunction-mediated unconventional cell death.
  • WB
  • Homo sapiens (Human)
Accumulation of 24S-OHC esters induced disruption of ER membrane integrity, resulting in release of ER luminal proteins into cytosol, in SH-SY5Y cells.a SH-SY5Y cells were treated with 50 μM 24S-OHC for 3 h. Cells were subjected to electron microscopy. Representative images are shown. Arrows indi... more
Accumulation of 24S-OHC esters induced disruption of ER membrane integrity, resulting in release of ER luminal proteins into cytosol, in SH-SY5Y cells.a SH-SY5Y cells were treated with 50 μM 24S-OHC for 3 h. Cells were subjected to electron microscopy. Representative images are shown. Arrows indicate intact rough ER. Arrowheads indicate broken-membrane ER structures. Scale bar, 0.2 μm. b, c Cells were pretreated with 5 μM F12511 for 15 min (b) or 20 μM MG132 for 30 min (c) and then exposed to 50 μM 24S-OHC for 3 h (b) or 6 h (c). Cells were then subjected to sequential detergent extraction using digitonin and NP-40 as indicated. Equal aliquots from each fraction were immunoblotted using antibodies as indicated. d, e Cells were pretreated with 5 μM TAK-243 for 30 min and then exposed to 50 μM 24S-OHC for 6 h. d Whole-cell lysates were subjected to immunoblotting with appropriate antibodies as indicated. e Band intensities were quantified by densitometric scanning, relative intensity being shown. n = 3, **P < 0.01, when compared with cells treated with vehicle or among 24S-OHC-treated groups less

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Connexin 43 plays an important role in the transformation of cholangiocytes with Clonochis sinensis excretory-secretory protein and N-nitrosodimethylamine.

In PLoS Negl Trop Dis on 1 April 2019 by Kim, E. M., Bae, Y. M., et al.

Fig.2.A
Fig.2.A showing Western Blotting in a Rattus norvegicus (Rat) sample from the publication: Connexin 43 plays an important role in the transformation of cholangiocytes with Clonochis sinensis excretory-secretory protein and N-nitrosodimethylamine.
  • WB
  • Rattus norvegicus (Rat)
Expression of E2F1, Ki-67, Cy-19, and Cox-2 in H69 cells after treatment with NDMA and ESP, assessed by western blotting.H69 cells were incubated with PBS (vehicle), NDMA, ESP, or both for 72 h. The cells were collected for protein extraction. A. The blots of each groups were run under same exper... more
Expression of E2F1, Ki-67, Cy-19, and Cox-2 in H69 cells after treatment with NDMA and ESP, assessed by western blotting.H69 cells were incubated with PBS (vehicle), NDMA, ESP, or both for 72 h. The cells were collected for protein extraction. A. The blots of each groups were run under same experimental conditions and the images were cropped from different parts of the same gels. B-E. Quantification of relative E2F1, Ki-67, Cy-19, and Cox-2 expression in each group. The each proteins level are indicated as normalization of the ratio of E2F1/Calnexin, Ki-67/Calnexin, Cy-19/Calnexin, and Cox-2/Calnexin. DATA represent the mean ± SE of five independent experiments.*P < 0.05, **P < 0.01 and ***P < 0.001 versus control. less

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Connexin 43 plays an important role in the transformation of cholangiocytes with Clonochis sinensis excretory-secretory protein and N-nitrosodimethylamine.

In PLoS Negl Trop Dis on 1 April 2019 by Kim, E. M., Bae, Y. M., et al.

Fig.3.A
Fig.3.A showing Western Blotting in a Rattus norvegicus (Rat) sample from the publication: Connexin 43 plays an important role in the transformation of cholangiocytes with Clonochis sinensis excretory-secretory protein and N-nitrosodimethylamine.
  • WB
  • Rattus norvegicus (Rat)
Expression of the gap-junction proteins connexin 26, connexin 32, and connexin 43 in H69 cells after treatment with NDMA and/or ESP, as determined by western blotting.H69 cells were incubated with either PBS (vehicle) or NDMA and/or ESP for 72 h, and the cells were collected for protein extractio... more
Expression of the gap-junction proteins connexin 26, connexin 32, and connexin 43 in H69 cells after treatment with NDMA and/or ESP, as determined by western blotting.H69 cells were incubated with either PBS (vehicle) or NDMA and/or ESP for 72 h, and the cells were collected for protein extraction. A. The blots of each groups were run under same experimental conditions and the images were cropped from different parts of the same gels. B-D. Quantification of relative Cx43 Cx26, and Cx32 expression in each group. The each proteins level are indicated as normalization of the ratio of Cx43/Calnexin, Cx26/Calnexin, and Cx32/Calnexin, and Cox-2/Calnexin. DATA represent the mean ± SE of five independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001 versus control. less

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LPAAT3 incorporates docosahexaenoic acid into skeletal muscle cell membranes and is upregulated by PPARδ activation.

In J Lipid Res on 1 February 2018 by Valentine, W. J., Tokuoka, S. M., et al.

Fig.4.B
Fig.4.B showing Western Blotting in a Mus musculus (House mouse) sample from the publication: LPAAT3 incorporates docosahexaenoic acid into skeletal muscle cell membranes and is upregulated by PPARδ activation.
  • WB
  • Mus musculus (House mouse)
LPAAT3 knockdown reduced DHA-containing PC and PE during satellite cell differentiation. Satellite cells were transfected with siRNAs and then cultured for 2 days in differentiation medium containing the fatty acid supplement (LA, AA, and DHA; 5 μM each). A, B: siRNA efficiency. LPAAT3-targeting ... more
LPAAT3 knockdown reduced DHA-containing PC and PE during satellite cell differentiation. Satellite cells were transfected with siRNAs and then cultured for 2 days in differentiation medium containing the fatty acid supplement (LA, AA, and DHA; 5 μM each). A, B: siRNA efficiency. LPAAT3-targeting siRNAs (siLPAAT3 #1 and #2) reduced LPAAT3 mRNA (A) and protein levels (B) compared with nontargeting siRNA (siCONTROL). A representative Western blot from three independent experiments is shown; calnexin was detected as a loading control. C–E: LPAAT3 knockdown decreased DHA-containing phospholipids. PC (C) and PE (D) species were measured by LC-MS. Several possible DHA-containing species were decreased by LPAAT3 knockdown. E: DHA-containing isomers of PC(38:6), PC(40:6), PE(38:6), and PE(40:6) were selectively reduced by the LPAAT3-targeting siRNAs. For each of these species, chromatographic peak areas were normalized to total PC or PE signals and expressed relative to the highest peak in the control siRNA sample. Acyl chain set compositions of the chromatographic peaks were determined by fragmentation in negative ion mode (Table 2, supplemental Fig. S1). All data represent the mean ± SEM of three or four independent experiments. *P < 0.05, control-siRNA versus LPAAT3-siRNA; one-way ANOVA (A) or two-way ANOVA (C–E). §Chromatographic peak containing mix of isomers. less

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LPAAT3 incorporates docosahexaenoic acid into skeletal muscle cell membranes and is upregulated by PPARδ activation.

In J Lipid Res on 1 February 2018 by Valentine, W. J., Tokuoka, S. M., et al.

Fig.2.E
Fig.2.E showing Western Blotting in a Mus musculus (House mouse) sample from the publication: LPAAT3 incorporates docosahexaenoic acid into skeletal muscle cell membranes and is upregulated by PPARδ activation.
  • WB
  • Mus musculus (House mouse)
LPAAT3 levels were upregulated during satellite cell differentiation, and expression was enhanced by fatty acid supplementation. Satellite cells were differentiated in low serum medium, and some media were supplemented with a fatty acid mixture of LA, AA, and DHA (5 μM each). mRNA expression leve... more
LPAAT3 levels were upregulated during satellite cell differentiation, and expression was enhanced by fatty acid supplementation. Satellite cells were differentiated in low serum medium, and some media were supplemented with a fatty acid mixture of LA, AA, and DHA (5 μM each). mRNA expression levels were measured by RT-PCR and protein levels were measured by Western blot. A: Images of satellite cells during differentiation. Satellite cells elongated and began fusing by day 1 and formed multinucleated myotubes by day 4. Scale bar = 100 μm. B–D: mRNA expression levels during satellite cell differentiation. B: LPAAT3 mRNA peaked at day 1 and expression was enhanced at day 4 by fatty acid supplementation. C: PPAR target genes UCP3 and PDK4 were also upregulated by fatty acid supplementation, suggesting possible PPAR activation. D: Components of the fatty acid mixture were applied individually (5 μM) during 4 days of differentiation. Either DHA or AA enhanced LPAAT3 expression. E: LPAAT3 protein increased during satellite cell differentiation and levels were enhanced by fatty acid supplementation at day 4. Representative Western blot from three independent experiments is shown; calnexin was detected as a loading control. RT-PCR data represent the mean ± SEM from three or four independent experiments. *P < 0.05, differentiated versus undifferentiated; #P < 0.05, fatty acid supplementation versus no supplementation; one–way ANOVA. less

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LPAAT3 incorporates docosahexaenoic acid into skeletal muscle cell membranes and is upregulated by PPARδ activation.

In J Lipid Res on 1 February 2018 by Valentine, W. J., Tokuoka, S. M., et al.

Fig.1.C
Fig.1.C showing Western Blotting in a Mus musculus (House mouse) sample from the publication: LPAAT3 incorporates docosahexaenoic acid into skeletal muscle cell membranes and is upregulated by PPARδ activation.
  • WB
  • Mus musculus (House mouse)
LPAAT3 was upregulated during differentiation of the C2C12 myoblast cell line. C2C12 cells were differentiated in low serum medium for 7 days, and then the myotubes were collected and differentiated for an additional 2 days. Undifferentiated cells were cultured in growth medium. A: Images of undi... more
LPAAT3 was upregulated during differentiation of the C2C12 myoblast cell line. C2C12 cells were differentiated in low serum medium for 7 days, and then the myotubes were collected and differentiated for an additional 2 days. Undifferentiated cells were cultured in growth medium. A: Images of undifferentiated and differentiated C2C12 cells are shown. Scale bar = 100 μm. B: LPAAT3 mRNA was increased during differentiation. Expression levels were measured by RT-PCR. Data are expressed as the mean ± SEM from three independent experiments. *P < 0.05; t-test. C: LPAAT3 protein levels increased during differentiation. LPAAT3 was detected by Western blot analyses. Calnexin was detected as a loading control. Representative blots of three independent experiments are shown. less

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Endosomal phosphatidylserine is critical for the YAP signalling pathway in proliferating cells.

In Nat Commun on 1 November 2017 by Matsudaira, T., Mukai, K., et al.

Fig.1.D
Fig.1.D showing Western Blotting in a Homo sapiens (Human) sample from the publication: Endosomal phosphatidylserine is critical for the YAP signalling pathway in proliferating cells.
  • WB
  • Homo sapiens (Human)
Identification of proteins proximal to PS in live cells. a Schematic illustration of biotinylation of proteins proximal to PS with GFP-BirA*-2xPH. b COS-1 cells stably expressing GFP-BirA*-2xPH were incubated with or without 50 µM biotin for 24 h. The cells were then fixed, permeabilized, and sta... more
Identification of proteins proximal to PS in live cells. a Schematic illustration of biotinylation of proteins proximal to PS with GFP-BirA*-2xPH. b COS-1 cells stably expressing GFP-BirA*-2xPH were incubated with or without 50 µM biotin for 24 h. The cells were then fixed, permeabilized, and stained for TfnR and biotin with Alexa594-streptavidin. c, d Lysates from cells in b were mixed with streptavidin-coupled magnetic beads. The proteins pulled down by the beads were blotted with streptavidin-HRP in c, or the indicated antibodies in d. Nuclei were stained with DAPI. Scale bars, 10 μm less

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Legionella effector Lpg1137 shuts down ER-mitochondria communication through cleavage of syntaxin 17.

In Nat Commun on 15 May 2017 by Arasaki, K., Mikami, Y., et al.

Fig.3.C
Fig.3.C showing Western Blotting in a Mus musculus (House mouse) sample from the publication: Legionella effector Lpg1137 shuts down ER-mitochondria communication through cleavage of syntaxin 17.
  • WB
  • Mus musculus (House mouse)
Lpg1137 is a serine protease localized in MAM/mitochondria.(a) HeLa-FcγRII cells were transfected with a plasmid encoding GFP (left) or GFP-Lpg1137 (right). At 24 h after transfection, cells were subjected to subcellular fractionation, and equal amounts of fractions were analysed by IB with the i... more
Lpg1137 is a serine protease localized in MAM/mitochondria.(a) HeLa-FcγRII cells were transfected with a plasmid encoding GFP (left) or GFP-Lpg1137 (right). At 24 h after transfection, cells were subjected to subcellular fractionation, and equal amounts of fractions were analysed by IB with the indicated antibodies. MS and MT denote microsomes and mitochondria, respectively. (b) HeLa-FcγRII cells were transfected with GFP-Lpg1137. At 4 h after transfection, DMSO (Vehicle), PMSF (1 mM) or MG132 (1 μM) was added to cells, and the cells were incubated for 20 h. Equal amounts of cell lysates were analysed by IB with the indicated antibodies. (c,d) HeLa-FcγRII cells were transfected with one of the indicated plasmids, and after 24 h equal amounts of lysates were analysed by IB with the (c) indicated antibodies. Alternatively, cells were fixed and stained with (d) an anti-Stx17 antibody. Scale bar, 5 μm. (e) His-Stx17 (0.2 μg) was incubated with MBP, MBP-Lpg1137 wild-type (WT) or MBP-Lpg1137 S68A (each 0.2 μg) for the indicated times at 37 °C. After incubation, samples were subjected to IB with antibodies against Stx17 and MBP. Uncropped images of blots are shown in Supplementary Fig. 7. less

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Legionella effector Lpg1137 shuts down ER-mitochondria communication through cleavage of syntaxin 17.

In Nat Commun on 15 May 2017 by Arasaki, K., Mikami, Y., et al.

Fig.3.B
Fig.3.B showing Western Blotting in a Mus musculus (House mouse) sample from the publication: Legionella effector Lpg1137 shuts down ER-mitochondria communication through cleavage of syntaxin 17.
  • WB
  • Mus musculus (House mouse)
Lpg1137 is a serine protease localized in MAM/mitochondria.(a) HeLa-FcγRII cells were transfected with a plasmid encoding GFP (left) or GFP-Lpg1137 (right). At 24 h after transfection, cells were subjected to subcellular fractionation, and equal amounts of fractions were analysed by IB with the i... more
Lpg1137 is a serine protease localized in MAM/mitochondria.(a) HeLa-FcγRII cells were transfected with a plasmid encoding GFP (left) or GFP-Lpg1137 (right). At 24 h after transfection, cells were subjected to subcellular fractionation, and equal amounts of fractions were analysed by IB with the indicated antibodies. MS and MT denote microsomes and mitochondria, respectively. (b) HeLa-FcγRII cells were transfected with GFP-Lpg1137. At 4 h after transfection, DMSO (Vehicle), PMSF (1 mM) or MG132 (1 μM) was added to cells, and the cells were incubated for 20 h. Equal amounts of cell lysates were analysed by IB with the indicated antibodies. (c,d) HeLa-FcγRII cells were transfected with one of the indicated plasmids, and after 24 h equal amounts of lysates were analysed by IB with the (c) indicated antibodies. Alternatively, cells were fixed and stained with (d) an anti-Stx17 antibody. Scale bar, 5 μm. (e) His-Stx17 (0.2 μg) was incubated with MBP, MBP-Lpg1137 wild-type (WT) or MBP-Lpg1137 S68A (each 0.2 μg) for the indicated times at 37 °C. After incubation, samples were subjected to IB with antibodies against Stx17 and MBP. Uncropped images of blots are shown in Supplementary Fig. 7. less

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Legionella effector Lpg1137 shuts down ER-mitochondria communication through cleavage of syntaxin 17.

In Nat Commun on 15 May 2017 by Arasaki, K., Mikami, Y., et al.

Fig.3.A
Fig.3.A showing Western Blotting in a Mus musculus (House mouse) sample from the publication: Legionella effector Lpg1137 shuts down ER-mitochondria communication through cleavage of syntaxin 17.
  • WB
  • Mus musculus (House mouse)
Lpg1137 is a serine protease localized in MAM/mitochondria.(a) HeLa-FcγRII cells were transfected with a plasmid encoding GFP (left) or GFP-Lpg1137 (right). At 24 h after transfection, cells were subjected to subcellular fractionation, and equal amounts of fractions were analysed by IB with the i... more
Lpg1137 is a serine protease localized in MAM/mitochondria.(a) HeLa-FcγRII cells were transfected with a plasmid encoding GFP (left) or GFP-Lpg1137 (right). At 24 h after transfection, cells were subjected to subcellular fractionation, and equal amounts of fractions were analysed by IB with the indicated antibodies. MS and MT denote microsomes and mitochondria, respectively. (b) HeLa-FcγRII cells were transfected with GFP-Lpg1137. At 4 h after transfection, DMSO (Vehicle), PMSF (1 mM) or MG132 (1 μM) was added to cells, and the cells were incubated for 20 h. Equal amounts of cell lysates were analysed by IB with the indicated antibodies. (c,d) HeLa-FcγRII cells were transfected with one of the indicated plasmids, and after 24 h equal amounts of lysates were analysed by IB with the (c) indicated antibodies. Alternatively, cells were fixed and stained with (d) an anti-Stx17 antibody. Scale bar, 5 μm. (e) His-Stx17 (0.2 μg) was incubated with MBP, MBP-Lpg1137 wild-type (WT) or MBP-Lpg1137 S68A (each 0.2 μg) for the indicated times at 37 °C. After incubation, samples were subjected to IB with antibodies against Stx17 and MBP. Uncropped images of blots are shown in Supplementary Fig. 7. less

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Legionella effector Lpg1137 shuts down ER-mitochondria communication through cleavage of syntaxin 17.

In Nat Commun on 15 May 2017 by Arasaki, K., Mikami, Y., et al.

Fig.6.B
Fig.6.B showing Western Blotting in a Mus musculus (House mouse) sample from the publication: Legionella effector Lpg1137 shuts down ER-mitochondria communication through cleavage of syntaxin 17.
  • WB
  • Mus musculus (House mouse)
Ectopic expression of Lpg1137 blocks autophagosome formation.(a) HeLa-FcγRII cells were transfected with a plasmid encoding GFP, GFP-Lpg1137 wild-type (WT) or GFP-Lpg1137 S68A. At 24 h after transfection, cells were incubated with EBSS (SV: left two columns) or EBSS plus 0.1 μM bafilomycin A1 (SV... more
Ectopic expression of Lpg1137 blocks autophagosome formation.(a) HeLa-FcγRII cells were transfected with a plasmid encoding GFP, GFP-Lpg1137 wild-type (WT) or GFP-Lpg1137 S68A. At 24 h after transfection, cells were incubated with EBSS (SV: left two columns) or EBSS plus 0.1 μM bafilomycin A1 (SV+bafilomycin A1, right two columns) for 2 h. After incubation, cells were fixed and stained with an anti-LC3 antibody. The bar graphs on the right show the ratio of the LC3 staining intensity of GFP-expressing to that of -nonexpressing cells. Values are means±s.d. (n=3). **P<0.001 as compared with GFP-Lgp1137 S68A. Scale bar, 5 μm. (b) HeLa-FcγRII cells were transfected with a plasmid encoding GFP or GFP-Lpg1137. At 24 h after transfection, cells were incubated with EBSS (SV)±0.1 μM bafilomycin A1 (Baf) for 2 h and then lysed. Equal amounts of the lysates were analysed by IB with the indicated antibodies. (c) HeLa-FcγRII cells expressing FLAG-DFCP1 (left two columns) or 3 × -FLAG-Atg14L (right two columns) were transfected with a plasmid encoding GFP, GFP-Lpg1137 WT or GFP-Lpg1137 S68A. At 24 h after transfection, cells were incubated with EBSS for 2 h, fixed and then stained with an anti-FALG antibody. The bar graphs shown at the bottom exhibit the ratio of the FLAG-DFCP1 or 3 × -FLAG-Atg14L staining intensity of GFP-expressing to that of -nonexpressing cells (50 cells each). Values are means±s.d. (n=3). **P<0.001 as compared with GFP-Lpg1137 S68A. Scale bar, 5 μm. (d) HeLa-FcγRII cells were transfected without (Mock) or with siRNA targeting Stx17 (Stx17 knockdown (KD)). At 48 h after transfection, cells were transfected with a plasmid encoding FLAG-DFCP1 or 3 × -FLAG-Atg14L, incubated for 24 h and then with EBSS for 2 h. The cells were fixed and stained with an anti-FLAG antibody. Scale bar, 5 μm. Uncropped images of blots are shown in Supplementary Fig. 7. less

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Legionella effector Lpg1137 shuts down ER-mitochondria communication through cleavage of syntaxin 17.

In Nat Commun on 15 May 2017 by Arasaki, K., Mikami, Y., et al.

Fig.1.C
Fig.1.C showing Western Blotting in a Mus musculus (House mouse) sample from the publication: Legionella effector Lpg1137 shuts down ER-mitochondria communication through cleavage of syntaxin 17.
  • WB
  • Mus musculus (House mouse)
Stx17 is degraded on Legionella infection.(a,b) HeLa-FcγRII cells were infected with (a) wild-type (WT) L. pneumophila or (b) one of the indicated strains at an MOI of 5. At 4 h after infection, cells were fixed and double stained with (a) an anti-Stx17 antibody and Hoechst 33342, or (b) an anti-... more
Stx17 is degraded on Legionella infection.(a,b) HeLa-FcγRII cells were infected with (a) wild-type (WT) L. pneumophila or (b) one of the indicated strains at an MOI of 5. At 4 h after infection, cells were fixed and double stained with (a) an anti-Stx17 antibody and Hoechst 33342, or (b) an anti-Legoinella serum. Asterisks and arrows indicate infected cells and Legionella, respectively. Scale bar, 5 μm. (c) HeLa-FcγRII cells were infected with the WT Legionella or a dotA mutant strain at an MOI of 50. At the indicated times, cell lysates were prepared, and equal amounts of proteins were subjected to SDS–PAGE followed by IB with antibodies against calnexin (CNX) and Stx17. (d) THP1 cells were treated as described in a except for infection at an MOI of 25, and double stained with antibodies against Stx17 and Legoinella. Scale bar, 5 μm. (e) Similar experiments as in c except that the indicated strains were used for infection. Uncropped images of blots are shown in Supplementary Fig. 7. less

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The Polyherbal Wattana Formula Displays Anti-Amyloidogenic Properties by Increasing α-Secretase Activities.

In PLoS One on 21 January 2017 by Htoo, H. H., Limsuvan, S., et al.

Fig.5.C
Fig.5.C showing Western Blotting from the publication: The Polyherbal Wattana Formula Displays Anti-Amyloidogenic Properties by Increasing α-Secretase Activities.
  • WB
WNF up-regulates ADAM10 and ADAM17 via two distinct mechanisms in human HEK293 cells.(A, B) HEK293 cells were treated for 36 hours without (control) or with WNF (100μg/ml) in DMEM/1% FBS. (A) ADAM10 (left panel), ADAM17 (right panel) as well as β-actin immunoreactivities were assayed by western b... more
WNF up-regulates ADAM10 and ADAM17 via two distinct mechanisms in human HEK293 cells.(A, B) HEK293 cells were treated for 36 hours without (control) or with WNF (100μg/ml) in DMEM/1% FBS. (A) ADAM10 (left panel), ADAM17 (right panel) as well as β-actin immunoreactivities were assayed by western blot. (B) ADAM10, ADAM17 as well as GAPDH mRNA levels were determined by real-time PCR. Black bars in histograms correspond to the densitometric analyses normalized with β-actin (A) or mRNA levels normalized with GAPDH (B), are expressed as a percentage of control (non-treated cells, white bars) and represent the means ± SE of 11 to 17 independent determinations. *p<0.0005; **p<0.0001; ns, non-statistically different. (C) HEK293 were incubated for 16 hours without (control) or with WNF (100μg/ml), homogenized and the sub-cellular distributions of endogenous ADAM10 (panels a and b), ADAM17 (panels c and d) as well as the ER marker calnexin (panel e) and the Golgi marker Golgi 58K protein (panel f) were analyzed by western blot after sucrose gradient fractionation. TGN, trans-golgi network; ER/PM, endoplasmic reticulum/plasma membrane. less

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Tetrahydrohyperforin Inhibits the Proteolytic Processing of Amyloid Precursor Protein and Enhances Its Degradation by Atg5-Dependent Autophagy.

In PLoS One on 27 August 2015 by Cavieres, V. A., Gonzalez, A., et al.

Fig.1.A
Fig.1.A showing Western Blotting from the publication: Tetrahydrohyperforin Inhibits the Proteolytic Processing of Amyloid Precursor Protein and Enhances Its Degradation by Atg5-Dependent Autophagy.
  • WB
IDN5706 reduces the levels of EDEM1 in a time-dependent manner.(A) H4 cells were left untreated or treated for different periods of time with 250 μM IDN5706, and subsequently analyzed by Western blotting with an EDEM1 antibody. The antibody specificity was validated on protein extracts from cells... more
IDN5706 reduces the levels of EDEM1 in a time-dependent manner.(A) H4 cells were left untreated or treated for different periods of time with 250 μM IDN5706, and subsequently analyzed by Western blotting with an EDEM1 antibody. The antibody specificity was validated on protein extracts from cells stably expressing either luciferase shRNA, used as positive control (shLuc), or EDEM1 shRNA (shEDEM1). Specific antibodies against ER proteins including Calnexin, Gp78 and Bip were tested. (B) Densitometric quantification of the levels of EDEM1 in cells left untreated or treated with IDN5706 for 16 h, as shown in A. Bars represent the mean ± SD of three independent experiments (***, P < 0.001). (C) H4 cells were either left untreated (lane 1) or transfected with a plasmid encoding HA-epitope-tagged EDEM1 (lanes 2 and 3). After 16 h cells were left untreated or treated with 250 μM IDN5706 for 8 h, and cellular extracts were subjected to SDS-PAGE followed by immunoblot with mouse antibody anti-HA-epitope. In A and C, Western blotting with antibody to β-actin was used as loading control. The position of molecular mass markers is indicated on the left. less

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Tagging strategies strongly affect the fate of overexpressed caveolin-1.

In Traffic on 1 April 2015 by Han, B., Tiwari, A., et al.

Fig.10.G
Fig.10.G showing Western Blotting from the publication: Tagging strategies strongly affect the fate of overexpressed caveolin-1.
  • WB
The affinity of overexpressed Cav1 for DRMs differs as a function of the tag. DRMs were isolated from (A) untransfected COS-7 cells or cells transiently transfected with (B) Cav1-myc, (C) P132L-myc, (D) Cav1-GFP, (E) P132L-GFP, (F) Cav1-mCherry or (G) P132L-mCherry. Fractions were analyzed by SDS... more
The affinity of overexpressed Cav1 for DRMs differs as a function of the tag. DRMs were isolated from (A) untransfected COS-7 cells or cells transiently transfected with (B) Cav1-myc, (C) P132L-myc, (D) Cav1-GFP, (E) P132L-GFP, (F) Cav1-mCherry or (G) P132L-mCherry. Fractions were analyzed by SDS–PAGE/western blotting. The levels of (H) overexpressed Cav1 and (I) endogenous Cav1 in each fraction were quantified by densitometry. The position of endogenous Cav1 in DRMs is indicated with a red line in (A–F). Bars represent mean ± SD for two independent experiments. less

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