CiteAb

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.A showing Western Blotting from the publication: Tagging strategies strongly affect the fate of overexpressed caveolin-1.

32 images found

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).

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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.

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Arf1-dependent LRBA recruitment to Rab4 endosomes is required for endolysosome homeostasis.

In The Journal of Cell Biology on 4 November 2024 by Szentgyörgyi, V., Lueck, L. M., et al.

Deleterious mutations in the lipopolysaccharide responsive beige-like anchor protein (LRBA) gene cause severe childhood immune dysregulation. The complexity of the symptoms involving multiple organs and the broad range of unpredictable clinical manifestations of LRBA deficiency complicate the choice of therapeutic interventions. Although LRBA has been linked to Rab11-dependent trafficking of the immune checkpoint protein CTLA-4, its precise cellular role remains elusive. We show that LRBA, however, only slightly colocalizes with Rab11. Instead, LRBA is recruited by members of the small GTPase Arf protein family to the TGN and to Rab4+ endosomes, where it controls intracellular traffic. In patient-derived fibroblasts, loss of LRBA led to defects in the endosomal pathway promoting the accumulation of enlarged endolysosomes and lysosome secretion. Thus, LRBA appears to regulate flow through the endosomal system on Rab4+ endosomes. Our data strongly suggest functions of LRBA beyond CTLA-4 trafficking and provide a conceptual framework to develop new therapies for LRBA deficiency.
© 2024 Szentgyörgyi et al.

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Small GTPase ActIvitY ANalyzing (SAIYAN) system: A method to detect GTPase activation in living cells.

In The Journal of Cell Biology on 7 October 2024 by Maeda, M., Arakawa, M., et al.

Small GTPases are essential in various cellular signaling pathways, and detecting their activation within living cells is crucial for understanding cellular processes. The current methods for detecting GTPase activation using fluorescent proteins rely on the interaction between the GTPase and its effector. Consequently, these methods are not applicable to factors, such as Sar1, where the effector also functions as a GTPase-activating protein. Here, we present a novel method, the Small GTPase ActIvitY ANalyzing (SAIYAN) system, for detecting the activation of endogenous small GTPases via fluorescent signals utilizing a split mNeonGreen system. We demonstrated Sar1 activation at the endoplasmic reticulum (ER) exit site and successfully detected its activation state in various cellular conditions. Utilizing the SAIYAN system in collagen-secreting cells, we discovered activated Sar1 localized both at the ER exit sites and ER-Golgi intermediate compartment (ERGIC) regions. Additionally, impaired collagen secretion confined the activated Sar1 at the ER exit sites, implying the importance of Sar1 activation through the ERGIC in collagen secretion.
© 2024 Maeda et al.

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Role of palmitoylation on the neuronal glycine transporter GlyT2.

In Journal of Neurochemistry on 1 September 2024 by Felipe, R., Sarmiento-Jiménez, J., et al.

The neuronal glycine transporter GlyT2 removes glycine from the synaptic cleft through active Na+, Cl-, and glycine cotransport contributing to the termination of the glycinergic signal as well as supplying substrate to the presynaptic terminal for the maintenance of the neurotransmitter content in synaptic vesicles. Patients with mutations in the human GlyT2 gene (SLC6A5), develop hyperekplexia or startle disease (OMIM 149400), characterized by hypertonia and exaggerated startle responses to trivial stimuli that may have lethal consequences in the neonates as a result of apnea episodes. Post-translational modifications in cysteine residues of GlyT2 are an aspect of structural interest we analyzed. Our study is compatible with a reversible and short-lived S-acylation in spinal cord membranes, detectable by biochemical and proteomics methods (acyl-Rac binding and IP-ABE) confirmed with positive and negative controls (palmitoylated and non-palmitoylated proteins). According to a short-lived modification, direct labeling using click chemistry was faint but mostly consistent. We have analyzed the physiological properties of a GlyT2 mutant lacking the cysteines with high prediction of palmitoylation and the mutant is less prone to be included in lipid rafts, an effect also observed upon treatment with the palmitoylation inhibitor 2-bromopalmitate. This work demonstrates there are determinants of lipid raft inclusion associated with the GlyT2 mutated cysteines, which are presumably modified by palmitoylation.
© 2024 The Author(s). Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.

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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.

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.

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.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.

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.

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.

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.

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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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.

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.

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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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.

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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