CiteAb

Fig.1.D showing Western Blotting in a Homo sapiens (Human) sample from the publication: ERLIN1/2 scaffolds bridge TMUB1 and RNF170 and restrict cholesterol esterification to regulate the secretory pathway.

Fig.5.E showing Western Blotting in a Mus musculus (House mouse) sample from the publication: Association of TRAIL receptor with phosphatase SHP-1 enables repressing T cell receptor signaling and T cell activation through inactivating Lck.

Fig.6.B showing Western Blotting in a Mus musculus (House mouse) sample from the publication: Association of TRAIL receptor with phosphatase SHP-1 enables repressing T cell receptor signaling and T cell activation through inactivating Lck.

Fig.1.E showing Western Blotting from the publication: Bone marrow-derived extracellular vesicles carry the TGF-β signal transducer Smad2 to preserve hematopoietic stem cells in mice.

Fig.3.D showing Western Blotting from the publication: Bone marrow-derived extracellular vesicles carry the TGF-β signal transducer Smad2 to preserve hematopoietic stem cells in mice.

Fig.5.B showing Western Blotting in a Homo sapiens (Human) sample from the publication: Selective immunocapture reveals neoplastic human mast cells secrete distinct microvesicle- and exosome-like populations of KIT-containing extracellular vesicles.

Fig.1.A showing Western Blotting in a Homo sapiens (Human) sample from the publication: Selective immunocapture reveals neoplastic human mast cells secrete distinct microvesicle- and exosome-like populations of KIT-containing extracellular vesicles.

Fig.1.F showing Western Blotting in a Homo sapiens (Human) sample from the publication: Selective immunocapture reveals neoplastic human mast cells secrete distinct microvesicle- and exosome-like populations of KIT-containing extracellular vesicles.

Fig.6.D showing Western Blotting in a Homo sapiens (Human) sample from the publication: Selective immunocapture reveals neoplastic human mast cells secrete distinct microvesicle- and exosome-like populations of KIT-containing extracellular vesicles.

Fig.6.E showing Western Blotting in a Homo sapiens (Human) sample from the publication: Selective immunocapture reveals neoplastic human mast cells secrete distinct microvesicle- and exosome-like populations of KIT-containing extracellular vesicles.

Fig.6.F showing Western Blotting in a Homo sapiens (Human) sample from the publication: Selective immunocapture reveals neoplastic human mast cells secrete distinct microvesicle- and exosome-like populations of KIT-containing extracellular vesicles.

Fig.3.A showing Western Blotting in a Homo sapiens (Human) sample from the publication: Selective immunocapture reveals neoplastic human mast cells secrete distinct microvesicle- and exosome-like populations of KIT-containing extracellular vesicles.

Fig.3.E showing Western Blotting in a Homo sapiens (Human) sample from the publication: Selective immunocapture reveals neoplastic human mast cells secrete distinct microvesicle- and exosome-like populations of KIT-containing extracellular vesicles.

Fig.9.B showing Western Blotting from the publication: Cerebellar Kv3.3 potassium channels activate TANK-binding kinase 1 to regulate trafficking of the cell survival protein Hax-1.

Fig.2.B showing Western Blotting from the publication: Cholesterol stimulates the cellular uptake of L-carnitine by the carnitine/organic cation transporter novel 2 (OCTN2).

Fig.1.E showing Western Blotting from the publication: The Urinary Excretion of Uromodulin is Regulated by the Potassium Channel ROMK.

Fig.1.D showing Western Blotting from the publication: The Urinary Excretion of Uromodulin is Regulated by the Potassium Channel ROMK.

Fig.6.F showing Western Blotting in a Homo sapiens (Human) sample from the publication: Quantifying exosome secretion from single cells reveals a modulatory role for GPCR signaling.

Fig.7.A showing Western Blotting in a Homo sapiens (Human) sample from the publication: HIV-1 Env associates with HLA-C free-chains at the cell membrane modulating viral infectivity.

19 images found

Glucosylceramide induced ectosomes propagate pathogenic α-synuclein in Parkinson’s disease

Preprint on BioRxiv : the Preprint Server for Biology on 16 February 2025 by Jacquemyn, J., Mariott, B., et al.

Intercellular transmission of α-synuclein contributes to Parkinson’s disease pathology. Yet, the mechanisms of α-synuclein spread are not fully understood. Here, we used live-cell microscopy to examine the impact of Parkinson’s disease associated lipid alterations on α-synuclein release. We discovered that increased glucosylceramides induce ectosome shedding from primary neurons, and from dopaminergic neurons derived from Parkinson’s disease patient iPSCs harboring mutations in GBA1 (N370S, L444P and W378G) and LRRK2 (G2019S and R1441H) compared to their isogenic control. We show that elevated glucosylceramide similarly increases vesicle release and uptake by other neurons in living mouse brains using 2-photon microscopy. Finally, we show that ectosomes are loaded with pathogenic α-synuclein and lead to the transmission of α-synuclein pathology to neighbouring neurons. These data reveal ectosomes as the predominant route for α-synuclein transmission that can only be appreciated by live-cell imaging technologies.

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Tau filaments are tethered within brain extracellular vesicles in Alzheimer's disease.

In Nature Neuroscience on 1 January 2025 by Fowler, S. L., Behr, T. S., et al.

The abnormal assembly of tau protein in neurons is a pathological hallmark of multiple neurodegenerative diseases, including Alzheimer's disease (AD). Assembled tau associates with extracellular vesicles (EVs) in the central nervous system of individuals with AD, which is linked to its clearance and prion-like propagation. However, the identities of the assembled tau species and EVs, as well as how they associate, are not known. Here, we combined quantitative mass spectrometry, cryo-electron tomography and single-particle cryo-electron microscopy to study brain EVs from individuals with AD. We found tau filaments composed mainly of truncated tau that were enclosed within EVs enriched in endo-lysosomal proteins. We observed multiple filament interactions, including with molecules that tethered filaments to the EV limiting membrane, suggesting selective packaging. Our findings will guide studies into the molecular mechanisms of EV-mediated secretion of assembled tau and inform the targeting of EV-associated tau as potential therapeutic and biomarker strategies for AD.
© 2024. Crown.

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Enhanced mGluR5 intracellular activity causes psychiatric alterations in Niemann Pick type C disease.

In Cell Death & Disease on 23 October 2024 by Toledano-Zaragoza, A., Enríquez-Zarralanga, V., et al.

Niemann-Pick disease Type C (NPC) is caused by mutations in the cholesterol transport protein NPC1 leading to the endolysosomal accumulation of the lipid and to psychiatric alterations. Using an NPC mouse model (Npc1nmf164) we show aberrant mGluR5 lysosomal accumulation and reduction at plasma membrane in NPC1 deficient neurons. This phenotype was induced in wild-type (wt) neurons by genetic and pharmacological NPC1 silencing. Extraction of cholesterol normalized mGluR5 distribution in NPC1-deficient neurons. Intracellular accumulation of mGluR5 was functionally active leading to enhanced mGluR-dependent long-term depression (mGluR-LTD) in Npc1nmf164 hippocampal slices. mGluR-LTD was lower or higher in Npc1nmf164 slices compared with wt when stimulated with non-membrane-permeable or membrane-permeable mGluR5 agonists, respectively. Oral treatment with the mGluR5 antagonist 2-chloro-4-((2,5-dimethyl-1-(4-(trifluoromethoxy)phenyl)-1H-imidazol-4-yl)ethynyl)pyridine (CTEP) reduced mGluR-LTD and ameliorated psychiatric anomalies in the Npc1nmf164 mice. Increased neuronal mGluR5 levels were found in an NPC patient. These results implicate mGluR5 alterations in NPC psychiatric condition and provide a new therapeutic strategy that might help patients suffering from this devastating disease.
© 2024. The Author(s).

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Small extracellular vesicles contain metals and transfer metal intercellularly.

In Journal of Extracellular Biology on 1 October 2024 by Purnianto, A., Mawal, C., et al.

Cells have developed a highly regulated system for the uptake, transport, utilization, storage, and export of metals, ensuring the maintenance of cellular homeostasis. Small extracellular vesicles (sEVs) function as a mechanism through which a cell can export its cargo and transfer it to recipient cells. However, in contrast to the other molecular cargo associated with sEVs, the metal content of sEVs is not well characterized. To address this gap in knowledge, we measured the levels of nine essential metals (copper, iron, zinc, manganese, magnesium, potassium, calcium, chromium, cobalt) and six non-essential metals (nickel, rubidium, titanium, aluminium, lithium, lead) in sEVs originating from multiple in vitro and ex vivo sources. Our findings reveal that, beyond containing redox-active essential metals and those involved in redox reactions, sEVs also exhibit the capability to export and transfer non-physiological, potentially toxic metals.
© 2024 The Author(s). Journal of Extracellular Biology published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.

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Maternal stress during pregnancy alters circulating small extracellular vesicles and enhances their targeting to the placenta and fetus.

In Biological Research on 28 September 2024 by Sánchez-Rubio, M., Abarzua-Catalan, L., et al.

Maternal psychological distress during pregnancy can negatively impact fetal development, resulting in long-lasting consequences for the offspring. These effects show a sex bias. The mechanisms whereby prenatal stress induces functional and/or structural changes in the placental-fetal unit remain poorly understood. Maternal circulating small extracellular vesicles (sEVs) are good candidates to act as "stress signals" in mother-to-fetus communication. Using a repetitive restraint-based rat model of prenatal stress, we examined circulating maternal sEVs under stress conditions and tested whether they could target placental-fetal tissues.
Our mild chronic maternal stress during pregnancy paradigm induced anhedonic-like behavior in pregnant dams and led to intrauterine growth restriction (IUGR), particularly in male fetuses and placentas. The concentration and cargo of maternal circulating sEVs changed under stress conditions. Specifically, there was a significant reduction in neuron-enriched proteins and a significant increase in astrocyte-enriched proteins in blood-borne sEVs from stressed dams. To study the effect of repetitive restraint stress on the biodistribution of maternal circulating sEVs in the fetoplacental unit, sEVs from pregnant dams exposed to stress or control protocol were labeled with DiR fluorescent die and injected into pregnant females previously exposed to control or stress protocol. Remarkably, maternal circulating sEVs target placental/fetal tissues and, under stress conditions, fetal tissues are more receptive to sEVs.
Our results suggest that maternal circulating sEVs can act as novel mediators/modulators of mother-to-fetus stress communication. Further studies are needed to identify placental/fetal cellular targets of maternal sEVs and characterize their contribution to stress-induced sex-specific placental and fetal changes.
© 2024. The Author(s).

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Association of TRAIL receptor with phosphatase SHP-1 enables repressing T cell receptor signaling and T cell activation through inactivating Lck.

In J Biomed Sci on 27 March 2024 by Chyuan, I. T., Liao, H. J., et al.

Fig.6.B

Fig.6.B showing Western Blotting in a Mus musculus (House mouse) sample from the publication: Association of TRAIL receptor with phosphatase SHP-1 enables repressing T cell receptor signaling and T cell activation through inactivating Lck.

SHP-1 knockdown abolished TRAIL-R-mediated inhibition on Lck phosphorylation, lipid raft recruitment and T cell activation. a Immunoblotting of phosphorylation of Lck and other proximal TCR signaling molecules (30 min), (b) immunoblotting of Lck and other proximal TCR signaling molecules in poole... more

SHP-1 knockdown abolished TRAIL-R-mediated inhibition on Lck phosphorylation, lipid raft recruitment and T cell activation. a Immunoblotting of phosphorylation of Lck and other proximal TCR signaling molecules (30 min), (b) immunoblotting of Lck and other proximal TCR signaling molecules in pooled raft fractions (30 min), (c) representative confocal images of co-localization of GM-1 with Lck (30 min), (d) flow cytometry of T cell activation markers CD69 and CD25 (24 h) (Scale bar, 4 µm), and (e) intracellular staining of IL-2 from murine splenic CD4.+ T cells transfected with scramble or SHP-1 siRNA, followed by stimulation with anti-CD3 (3 µg/mL) and anti-CD28 (2 µg/mL) Abs in the presence/absence of TRAIL (0–1 µg/mL) for 24 h. Quantification of phosphorylated protein levels are shown at the bottom of the panel. Data d–e represent analyses from three independent experiments and statistics determined using Mann–Whitney U test. NS, not significant; *** p < 0.001. f Model of TRAIL-R/SHP-1 repressing TCR signaling and T cell activation through dephosphorylation of Lck at Y394. Upon TCR ligation, Lck binds to, and phosphorylates TCR and Zap70 to form an immunological synapse in the lipid raft, which transduces proximal TCR signaling downstream, resulting in T cell activation (Left). TRAIL engagement reduces Lck (Y394) phosphorylation along with the increased phosphorylated SHP-1 in the TRAIL-R/SHP-1/Lck complex, leading to the dissociation of the co-receptor-Lck complex from the lipid raft, which in turn limits downstream TCR signaling to restrain T cell activation (Right) less

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Selective immunocapture reveals neoplastic human mast cells secrete distinct microvesicle- and exosome-like populations of KIT-containing extracellular vesicles.

In J Extracell Vesicles on 1 September 2022 by Pfeiffer, A., Petersen, J. D., et al.

Fig.5.B

Fig.5.B showing Western Blotting in a Homo sapiens (Human) sample from the publication: Selective immunocapture reveals neoplastic human mast cells secrete distinct microvesicle- and exosome-like populations of KIT-containing extracellular vesicles.

Mast cells produce microvesicle (P15)‐ and exosome (P120)‐like KIT‐EVs. (A) Schematic of KIT immunoprecipitation from P15 and P120 EV populations. (B) KIT‐EVs were purified from P15 or P120 EVs (8.9 × 109 EVs) by immunoprecipitation with an antibody against N‐terminal KIT. EVs in the flow‐through... more

Mast cells produce microvesicle (P15)‐ and exosome (P120)‐like KIT‐EVs. (A) Schematic of KIT immunoprecipitation from P15 and P120 EV populations. (B) KIT‐EVs were purified from P15 or P120 EVs (8.9 × 109 EVs) by immunoprecipitation with an antibody against N‐terminal KIT. EVs in the flow‐through were captured with CD9‐conjugated beads. Unconjugated beads and a mix of P15 and P120 vesicles were used as a negative control. Cell lysates (30 μg) and EV eluates were analyzed by Western blotting with the indicated antibodies. Representative blots are shown. *, indicates bands arising from protein G. (C) Relative signal intensities of protein markers shown in (B) were quantified and normalized to signals in P15 KIT‐EVs. Data are shown as mean ± S.D. from three independent experiments. Proteins enriched in P120 and P15 KIT‐EVs are highlighted in orange and blue, respectively. Ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 (unpaired Student t‐test). (D) The concentration of P15 and P120 EVs before and after KIT immunoprecipitation shown in (B) was measured by nanoparticle tracking. The representative profile shows the distribution of particle numbers for a range of particle sizes averaged from five workflow repetitions. The sizes (nm) of the most abundant particles in P15 and P120 are indicated. The percentage of KIT‐EVs was estimated by determining the particle count before and after KIT‐EV capturing. FT, flow through. (E) After SILAC labelling of HMC‐1.1 cells with heavy and light amino acids, EVs were isolated from the cell culture supernatant (SN) by ultracentrifugation as P15 and P120 EV pellets. EV suspensions were further cleaned by size exclusion chromatography (SEC) before performing KIT immunocapturing on each EV pool. Heavy‐labeled KIT P15 EVs were mixed with light‐labelled KIT P120 EVs (and vice versa) and processed for quantitative mass spectrometry as inverse duplicates. (F) The enrichment of exemplary proteins (presented as log2 fold change H/L) determined by SILAC mass spectrometry in P15 KIT‐EVs compared to P120 KIT‐EVs is shown. Data are presented as the mean from two mass spectrometry pairs ± S.D. The red line indicates the 2‐fold enrichment threshold. Proteins highly enriched in P15 KIT‐EVs are indicated in blue. The ‐Log T‐test p‐value is shown above each bar. ‐Log T‐test p‐values > 1.3 indicate significance. (G) Illustration of P15 and P120 KIT‐EVs summarizing the association of EV markers with respective vesicle types. Protein markers highly associated with either P15 (microvesicle‐like) or P120 (exosome‐like) EVs are highlighted in red less

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Selective immunocapture reveals neoplastic human mast cells secrete distinct microvesicle- and exosome-like populations of KIT-containing extracellular vesicles.

In J Extracell Vesicles on 1 September 2022 by Pfeiffer, A., Petersen, J. D., et al.

Fig.1.A

Fig.1.A showing Western Blotting in a Homo sapiens (Human) sample from the publication: Selective immunocapture reveals neoplastic human mast cells secrete distinct microvesicle- and exosome-like populations of KIT-containing extracellular vesicles.

HMC‐1‐derived EVs contain KIT and canonical EV protein markers. (A, B) Detection of canonical EV marker (A) and mast cell marker proteins (B) as well as calreticulin as an exclusion marker in HMC‐1.1 and HMC‐1.2 cells and secreted EVs. 30 μg of protein of cell or EV lysates were separated by SDS‐... more

HMC‐1‐derived EVs contain KIT and canonical EV protein markers. (A, B) Detection of canonical EV marker (A) and mast cell marker proteins (B) as well as calreticulin as an exclusion marker in HMC‐1.1 and HMC‐1.2 cells and secreted EVs. 30 μg of protein of cell or EV lysates were separated by SDS‐PAGE, blotted and probed with the indicated antibodies. (C) HMC‐1.1‐derived EVs were separated from soluble protein by size exclusion chromatography (SEC; qEVoriginal70) and eluted as pooled fractions F7‐F11, followed by filter concentration. Cell (30 μg) and EV lysates (15 μg) were analyzed by immunoblotting with the indicated antibodies. The dashed line indicates the samples were run on the same blot, but not in contiguous lanes. (D) A representative graph showing the concentration and size measurement of SEC‐isolated EVs (C) by nanoparticle tracking. The average profile of five workflow repetitions is displayed. (E) Workflow of the immunocapture of EV subpopulations. EVs are bulk‐isolated from pre‐cleared cell culture media by PEG‐precipitation and incubated with antibody‐conjugated beads allowing for immunocapture of specific EV subpopulations. (F) Representative Western blot of CD9‐, CD81‐ and CD63‐immunocaptured EVs. Each bead capture was performed from an equal number of HMC‐1.1‐derived EVs (1.9 × 1010 EVs). Unconjugated beads served as a negative control. The presence of canonical EV protein markers as well as KIT in the EV subpopulations were determined by immunoblotting. *, signal from previous probing with a flotillin‐1 antibody less

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Selective immunocapture reveals neoplastic human mast cells secrete distinct microvesicle- and exosome-like populations of KIT-containing extracellular vesicles.

In J Extracell Vesicles on 1 September 2022 by Pfeiffer, A., Petersen, J. D., et al.

Fig.1.F

Fig.1.F showing Western Blotting in a Homo sapiens (Human) sample from the publication: Selective immunocapture reveals neoplastic human mast cells secrete distinct microvesicle- and exosome-like populations of KIT-containing extracellular vesicles.

HMC‐1‐derived EVs contain KIT and canonical EV protein markers. (A, B) Detection of canonical EV marker (A) and mast cell marker proteins (B) as well as calreticulin as an exclusion marker in HMC‐1.1 and HMC‐1.2 cells and secreted EVs. 30 μg of protein of cell or EV lysates were separated by SDS‐... more

HMC‐1‐derived EVs contain KIT and canonical EV protein markers. (A, B) Detection of canonical EV marker (A) and mast cell marker proteins (B) as well as calreticulin as an exclusion marker in HMC‐1.1 and HMC‐1.2 cells and secreted EVs. 30 μg of protein of cell or EV lysates were separated by SDS‐PAGE, blotted and probed with the indicated antibodies. (C) HMC‐1.1‐derived EVs were separated from soluble protein by size exclusion chromatography (SEC; qEVoriginal70) and eluted as pooled fractions F7‐F11, followed by filter concentration. Cell (30 μg) and EV lysates (15 μg) were analyzed by immunoblotting with the indicated antibodies. The dashed line indicates the samples were run on the same blot, but not in contiguous lanes. (D) A representative graph showing the concentration and size measurement of SEC‐isolated EVs (C) by nanoparticle tracking. The average profile of five workflow repetitions is displayed. (E) Workflow of the immunocapture of EV subpopulations. EVs are bulk‐isolated from pre‐cleared cell culture media by PEG‐precipitation and incubated with antibody‐conjugated beads allowing for immunocapture of specific EV subpopulations. (F) Representative Western blot of CD9‐, CD81‐ and CD63‐immunocaptured EVs. Each bead capture was performed from an equal number of HMC‐1.1‐derived EVs (1.9 × 1010 EVs). Unconjugated beads served as a negative control. The presence of canonical EV protein markers as well as KIT in the EV subpopulations were determined by immunoblotting. *, signal from previous probing with a flotillin‐1 antibody less

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Selective immunocapture reveals neoplastic human mast cells secrete distinct microvesicle- and exosome-like populations of KIT-containing extracellular vesicles.

In J Extracell Vesicles on 1 September 2022 by Pfeiffer, A., Petersen, J. D., et al.

Fig.6.D

Fig.6.D showing Western Blotting in a Homo sapiens (Human) sample from the publication: Selective immunocapture reveals neoplastic human mast cells secrete distinct microvesicle- and exosome-like populations of KIT-containing extracellular vesicles.

The protein loading of KIT‐EVs shifts among vesicle subtypes after cell treatment with SMase inhibitors. (A) Viability assessment of HMC‐1.1 cells at the end of the 16 h treatment with DMSO (vehicle control), 10 μM GW4869 (n‐SMase inhibitor) or 10 μM amitriptyline (a‐SMase inhibitor) to control f... more

The protein loading of KIT‐EVs shifts among vesicle subtypes after cell treatment with SMase inhibitors. (A) Viability assessment of HMC‐1.1 cells at the end of the 16 h treatment with DMSO (vehicle control), 10 μM GW4869 (n‐SMase inhibitor) or 10 μM amitriptyline (a‐SMase inhibitor) to control for potential drug‐induced cell death. Data are presented as mean ± S.D. from three independent experiments, normalized to the control. (B, C) EVs released by control‐ or inhibitor‐treated HMC‐1.1 cells were pelleted as P15 (B) or P120 (C) EVs and evaluated by nanoparticle tracking. The sum of P15 (B) or P120 (C) EVs (ranging from 0 to 250 nm) released under each condition was plotted. Representative data are shown as mean ± S.D. from five repetitions. Ns, not significant; **, 0.0011; ****p < 0.0001 (two‐tailed, unpaired Student t‐test). (D) Total lysates (30 μg) of DMSO‐ or inhibitor‐treated cells were analyzed by immunoblotting with the indicated antibodies. (E,F) KIT‐EVs were captured with an antibody against N‐terminal KIT from P15 (3.4 × 108 EVs) (E) or P120 (2.6 × 109 EVs) (F) vesicles released by control, GW4869‐ or amitriptyline‐treated cells. Total KIT‐EV lysates were evaluated by Western blotting; representative blots are shown. (G,H) Signal intensities of proteins in KIT‐EVs (E,F) were quantified and normalized to the DMSO control. Up‐ or down‐regulation (indicated by arrows) of protein content in P15 and P120 KIT‐EVs in response to GW4869 (G) or amitriptyline (H) is displayed in Volcano graphs (n = 3). The q‐value is the FDR‐adjusted p‐value, and the dashed line indicates the significance threshold. In D, E and F, dashed lines imply lanes from the same blot that were not loaded contiguously less

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Selective immunocapture reveals neoplastic human mast cells secrete distinct microvesicle- and exosome-like populations of KIT-containing extracellular vesicles.

In J Extracell Vesicles on 1 September 2022 by Pfeiffer, A., Petersen, J. D., et al.

Fig.6.E

Fig.6.E showing Western Blotting in a Homo sapiens (Human) sample from the publication: Selective immunocapture reveals neoplastic human mast cells secrete distinct microvesicle- and exosome-like populations of KIT-containing extracellular vesicles.

The protein loading of KIT‐EVs shifts among vesicle subtypes after cell treatment with SMase inhibitors. (A) Viability assessment of HMC‐1.1 cells at the end of the 16 h treatment with DMSO (vehicle control), 10 μM GW4869 (n‐SMase inhibitor) or 10 μM amitriptyline (a‐SMase inhibitor) to control f... more

The protein loading of KIT‐EVs shifts among vesicle subtypes after cell treatment with SMase inhibitors. (A) Viability assessment of HMC‐1.1 cells at the end of the 16 h treatment with DMSO (vehicle control), 10 μM GW4869 (n‐SMase inhibitor) or 10 μM amitriptyline (a‐SMase inhibitor) to control for potential drug‐induced cell death. Data are presented as mean ± S.D. from three independent experiments, normalized to the control. (B, C) EVs released by control‐ or inhibitor‐treated HMC‐1.1 cells were pelleted as P15 (B) or P120 (C) EVs and evaluated by nanoparticle tracking. The sum of P15 (B) or P120 (C) EVs (ranging from 0 to 250 nm) released under each condition was plotted. Representative data are shown as mean ± S.D. from five repetitions. Ns, not significant; **, 0.0011; ****p < 0.0001 (two‐tailed, unpaired Student t‐test). (D) Total lysates (30 μg) of DMSO‐ or inhibitor‐treated cells were analyzed by immunoblotting with the indicated antibodies. (E,F) KIT‐EVs were captured with an antibody against N‐terminal KIT from P15 (3.4 × 108 EVs) (E) or P120 (2.6 × 109 EVs) (F) vesicles released by control, GW4869‐ or amitriptyline‐treated cells. Total KIT‐EV lysates were evaluated by Western blotting; representative blots are shown. (G,H) Signal intensities of proteins in KIT‐EVs (E,F) were quantified and normalized to the DMSO control. Up‐ or down‐regulation (indicated by arrows) of protein content in P15 and P120 KIT‐EVs in response to GW4869 (G) or amitriptyline (H) is displayed in Volcano graphs (n = 3). The q‐value is the FDR‐adjusted p‐value, and the dashed line indicates the significance threshold. In D, E and F, dashed lines imply lanes from the same blot that were not loaded contiguously less

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Selective immunocapture reveals neoplastic human mast cells secrete distinct microvesicle- and exosome-like populations of KIT-containing extracellular vesicles.

In J Extracell Vesicles on 1 September 2022 by Pfeiffer, A., Petersen, J. D., et al.

Fig.6.F

Fig.6.F showing Western Blotting in a Homo sapiens (Human) sample from the publication: Selective immunocapture reveals neoplastic human mast cells secrete distinct microvesicle- and exosome-like populations of KIT-containing extracellular vesicles.

The protein loading of KIT‐EVs shifts among vesicle subtypes after cell treatment with SMase inhibitors. (A) Viability assessment of HMC‐1.1 cells at the end of the 16 h treatment with DMSO (vehicle control), 10 μM GW4869 (n‐SMase inhibitor) or 10 μM amitriptyline (a‐SMase inhibitor) to control f... more

The protein loading of KIT‐EVs shifts among vesicle subtypes after cell treatment with SMase inhibitors. (A) Viability assessment of HMC‐1.1 cells at the end of the 16 h treatment with DMSO (vehicle control), 10 μM GW4869 (n‐SMase inhibitor) or 10 μM amitriptyline (a‐SMase inhibitor) to control for potential drug‐induced cell death. Data are presented as mean ± S.D. from three independent experiments, normalized to the control. (B, C) EVs released by control‐ or inhibitor‐treated HMC‐1.1 cells were pelleted as P15 (B) or P120 (C) EVs and evaluated by nanoparticle tracking. The sum of P15 (B) or P120 (C) EVs (ranging from 0 to 250 nm) released under each condition was plotted. Representative data are shown as mean ± S.D. from five repetitions. Ns, not significant; **, 0.0011; ****p < 0.0001 (two‐tailed, unpaired Student t‐test). (D) Total lysates (30 μg) of DMSO‐ or inhibitor‐treated cells were analyzed by immunoblotting with the indicated antibodies. (E,F) KIT‐EVs were captured with an antibody against N‐terminal KIT from P15 (3.4 × 108 EVs) (E) or P120 (2.6 × 109 EVs) (F) vesicles released by control, GW4869‐ or amitriptyline‐treated cells. Total KIT‐EV lysates were evaluated by Western blotting; representative blots are shown. (G,H) Signal intensities of proteins in KIT‐EVs (E,F) were quantified and normalized to the DMSO control. Up‐ or down‐regulation (indicated by arrows) of protein content in P15 and P120 KIT‐EVs in response to GW4869 (G) or amitriptyline (H) is displayed in Volcano graphs (n = 3). The q‐value is the FDR‐adjusted p‐value, and the dashed line indicates the significance threshold. In D, E and F, dashed lines imply lanes from the same blot that were not loaded contiguously less

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Selective immunocapture reveals neoplastic human mast cells secrete distinct microvesicle- and exosome-like populations of KIT-containing extracellular vesicles.

In J Extracell Vesicles on 1 September 2022 by Pfeiffer, A., Petersen, J. D., et al.

Fig.3.A

Fig.3.A showing Western Blotting in a Homo sapiens (Human) sample from the publication: Selective immunocapture reveals neoplastic human mast cells secrete distinct microvesicle- and exosome-like populations of KIT-containing extracellular vesicles.

KIT‐captured EVs reveal distinct EV marker profiles compared to CD9‐enriched vesicles. (A) Comparison of KIT‐ vs. CD9‐captured EV populations. Each EV immunoprecipitation was performed from an equal number of EVs (3.5 × 1010 EVs), and the protein content was assessed by immunoblotting. Unconjugat... more

KIT‐captured EVs reveal distinct EV marker profiles compared to CD9‐enriched vesicles. (A) Comparison of KIT‐ vs. CD9‐captured EV populations. Each EV immunoprecipitation was performed from an equal number of EVs (3.5 × 1010 EVs), and the protein content was assessed by immunoblotting. Unconjugated beads served as a negative control. KIT‐EV immunocapture was performed with an antibody against N‐terminal KIT. Representative blots are shown. Signal intensities of precipitated proteins were normalized to the adjusted shown input (the input equalled 8.9% of the starting material of an IP) and indicated below each blot. Cell lysates (30 μg) were loaded in the first lane. (B) Quantification of protein signal intensities (A) in CD9‐EVs compared to KIT‐EVs. Data are represented as mean ± S.D. from three independent experiments. Proteins that are strongly represented in KIT‐EVs and reduced in CD9‐captured EVs are highlighted in blue. (C) Correlation plot representing the relative enrichment of probed proteins in KIT‐ vs. CD9‐EVs. (D) Illustration of the isolation procedure of KIT‐positive (KIT(+)) and KIT‐depleted (KIT(‐)) EVs. EVs were first immunocaptured by KIT‐conjugated beads and total eluates were analyzed. The flow‐through, containing KIT‐depleted EVs, was further processed by immunocapture with CD9‐conjugated beads yielding CD9‐positive/KIT‐negative (CD9(+)/KIT(‐)) EVs. (E) Representative immunoblot of bead‐captured KIT‐positive (second lane) and CD9(+)/KIT(‐) EVs from the flow‐through (third lane). Unconjugated beads were used as a negative control (first lane). KIT immunocapture was performed with an antibody against N‐terminal KIT from 2.4 × 1010 EVs. FT, flow‐through. (F) Signal intensities of protein markers (E) were quantified and are represented as mean ± S.D. from three independent experiments and normalized to KIT(+) EVs. Protein markers largely absent from KIT‐depleted EVs are highlighted in blue. Ns, not significant; *p < 0.05; **p <0.01; ***p < 0.001; ****p < 0.0001 (unpaired Student t‐test) less

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Selective immunocapture reveals neoplastic human mast cells secrete distinct microvesicle- and exosome-like populations of KIT-containing extracellular vesicles.

In J Extracell Vesicles on 1 September 2022 by Pfeiffer, A., Petersen, J. D., et al.

Fig.3.E

Fig.3.E showing Western Blotting in a Homo sapiens (Human) sample from the publication: Selective immunocapture reveals neoplastic human mast cells secrete distinct microvesicle- and exosome-like populations of KIT-containing extracellular vesicles.

KIT‐captured EVs reveal distinct EV marker profiles compared to CD9‐enriched vesicles. (A) Comparison of KIT‐ vs. CD9‐captured EV populations. Each EV immunoprecipitation was performed from an equal number of EVs (3.5 × 1010 EVs), and the protein content was assessed by immunoblotting. Unconjugat... more

KIT‐captured EVs reveal distinct EV marker profiles compared to CD9‐enriched vesicles. (A) Comparison of KIT‐ vs. CD9‐captured EV populations. Each EV immunoprecipitation was performed from an equal number of EVs (3.5 × 1010 EVs), and the protein content was assessed by immunoblotting. Unconjugated beads served as a negative control. KIT‐EV immunocapture was performed with an antibody against N‐terminal KIT. Representative blots are shown. Signal intensities of precipitated proteins were normalized to the adjusted shown input (the input equalled 8.9% of the starting material of an IP) and indicated below each blot. Cell lysates (30 μg) were loaded in the first lane. (B) Quantification of protein signal intensities (A) in CD9‐EVs compared to KIT‐EVs. Data are represented as mean ± S.D. from three independent experiments. Proteins that are strongly represented in KIT‐EVs and reduced in CD9‐captured EVs are highlighted in blue. (C) Correlation plot representing the relative enrichment of probed proteins in KIT‐ vs. CD9‐EVs. (D) Illustration of the isolation procedure of KIT‐positive (KIT(+)) and KIT‐depleted (KIT(‐)) EVs. EVs were first immunocaptured by KIT‐conjugated beads and total eluates were analyzed. The flow‐through, containing KIT‐depleted EVs, was further processed by immunocapture with CD9‐conjugated beads yielding CD9‐positive/KIT‐negative (CD9(+)/KIT(‐)) EVs. (E) Representative immunoblot of bead‐captured KIT‐positive (second lane) and CD9(+)/KIT(‐) EVs from the flow‐through (third lane). Unconjugated beads were used as a negative control (first lane). KIT immunocapture was performed with an antibody against N‐terminal KIT from 2.4 × 1010 EVs. FT, flow‐through. (F) Signal intensities of protein markers (E) were quantified and are represented as mean ± S.D. from three independent experiments and normalized to KIT(+) EVs. Protein markers largely absent from KIT‐depleted EVs are highlighted in blue. Ns, not significant; *p < 0.05; **p <0.01; ***p < 0.001; ****p < 0.0001 (unpaired Student t‐test) less

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Cerebellar Kv3.3 potassium channels activate TANK-binding kinase 1 to regulate trafficking of the cell survival protein Hax-1.

In Nat Commun on 19 March 2021 by Zhang, Y., Varela, L., et al.

Fig.9.B

Fig.9.B showing Western Blotting from the publication: Cerebellar Kv3.3 potassium channels activate TANK-binding kinase 1 to regulate trafficking of the cell survival protein Hax-1.

The Kv3.3 G592R mutation promotes release of exosomes into the extracellular space.a Representative transmission electron microscopy photomicrographs of low density fraction (LDF) and intermediate-density fraction (IDF) extracellular vesicles (EVs) isolated from the cerebellum of a wild type mous... more

The Kv3.3 G592R mutation promotes release of exosomes into the extracellular space.a Representative transmission electron microscopy photomicrographs of low density fraction (LDF) and intermediate-density fraction (IDF) extracellular vesicles (EVs) isolated from the cerebellum of a wild type mouse. Scale bar: 500 nm. Images are representative of ten images taken of each of the sets analyzed. b Representative Western blot analyses of LDF EVs (L) and IDF EVs (I) compared to the cerebellar homogenate (BH). c Nanotrack analysis (NTA) of the hydrodynamic diameter of the EVs found in LDFs and IDFs. The curve is normalized to the mode of each distribution (five independent isolations. d Quantification of particles in LDFs and IDFs by NTA that are found within the size bins shown in the graph. Data are plotted as percentage of total number of EVs. Five independent isolations. e Representative Western blot analyses of EVs isolated from cerebella of wild type and G592R Kv3.3 mutant mice. LDFs (L) and IDFs (I) were isolated from cerebella of two mice of the same genotype and these were combined and tested for the markers shown. Two independent experiments are shown in the blot. f–h quantification of data in e. The densitometric quantification was performed on three independent experiments, representative of six mice per genotype. BCA protein assay (i) and total particle count as estimated by NTA (j) of LDFs and IDFs in wild type and G592R Kv3.3 mice. Values were normalized to the cerebellum wet weight (five independent isolations for wild type and three independent isolations for mutant mice, corresponding to ten and six mice, respectively. All data are shown as a mean ± SEM. Statistical test used in all figures: Two-Way ANOVA with Sidak’s multiple comparisons test). Source data and uncropped Western blots are provided as a Source Data file. less

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The Urinary Excretion of Uromodulin is Regulated by the Potassium Channel ROMK.

In Sci Rep on 20 December 2019 by Schiano, G., Glaudemans, B., et al.

Fig.1.E

Fig.1.E showing Western Blotting from the publication: The Urinary Excretion of Uromodulin is Regulated by the Potassium Channel ROMK.

Histological characterization, transcript analysis and uromodulin processing in Kcnj1−/− mice. (a) Haematoxylin and eosin (H&E) staining of 12 weeks Kcnj1+/+ and Kcnj1−/− kidneys. Scale bars: low magnification - 2 mm, high magnification - 100 μm (b) Quantitative PCR analysis of tubular marker gen... more

Histological characterization, transcript analysis and uromodulin processing in Kcnj1−/− mice. (a) Haematoxylin and eosin (H&E) staining of 12 weeks Kcnj1+/+ and Kcnj1−/− kidneys. Scale bars: low magnification - 2 mm, high magnification - 100 μm (b) Quantitative PCR analysis of tubular marker genes in Kcnj1−/− kidneys. Glom (Glomerulus): Nphs2 (Podocin); PT (Proximal Tubule): Slc5a2 - Sodium/glucose cotransporter 2 (SGLT2), Aqp1 - Aquaporin 1; TAL (Thick Ascending Limb): Slc12a1 - Na/K/Cl cotransporter 2 (NKCC2), Umod - Uromodulin; DCT (Distal Convoluted Tubule): Slc12a3 - Na/Cl cotransporter (NCC), Pvalb - Parvalbumin; CNT/CD (Connecting Tubule/Collecting Duct): Aqp2 - Aquaporin 2, Scnn1b - Sodium channel epithelial 1 beta subunit (β-ENaC), Scnn1g - Sodium channel epithelial 1 gamma subunit (γ-ENaC); Cldn16 - Claudin 16 and Clcnkb (Cl channel Kb) are localized in both TAL and DCT. (c) Representative Western blot analysis on whole kidney lysates of 12 weeks Kcnj1+/+ and Kcnj1−/− mice. β-actin was used as a loading control. (d) Representative Western blot analysis on kidney membrane fractions of 12 weeks Kcnj1+/+ and Kcnj1−/− mice. Flotillin-1 was used as a loading control, whereas GAPDH was used to test cytosolic contamination. (e) Representative Western blot analysis on kidney cytosolic fractions of 12 weeks Kcnj1+/+ and Kcnj1−/− mice. GAPDH was used as a loading control, whereas Flotillin-1 was used to test membrane contamination. (f) Immunofluorescence analysis for UMOD (green) and NKCC2 (red) in Kcnj1+/+ and Kcnj1−/− serial kidney sections. Scale bars: low magnification - 50 μm, high magnification - 15 μm. Full length blot images can be found in Supplementary Figure S2. less

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The Urinary Excretion of Uromodulin is Regulated by the Potassium Channel ROMK.

In Sci Rep on 20 December 2019 by Schiano, G., Glaudemans, B., et al.

Fig.1.D

Fig.1.D showing Western Blotting from the publication: The Urinary Excretion of Uromodulin is Regulated by the Potassium Channel ROMK.

Histological characterization, transcript analysis and uromodulin processing in Kcnj1−/− mice. (a) Haematoxylin and eosin (H&E) staining of 12 weeks Kcnj1+/+ and Kcnj1−/− kidneys. Scale bars: low magnification - 2 mm, high magnification - 100 μm (b) Quantitative PCR analysis of tubular marker gen... more

Histological characterization, transcript analysis and uromodulin processing in Kcnj1−/− mice. (a) Haematoxylin and eosin (H&E) staining of 12 weeks Kcnj1+/+ and Kcnj1−/− kidneys. Scale bars: low magnification - 2 mm, high magnification - 100 μm (b) Quantitative PCR analysis of tubular marker genes in Kcnj1−/− kidneys. Glom (Glomerulus): Nphs2 (Podocin); PT (Proximal Tubule): Slc5a2 - Sodium/glucose cotransporter 2 (SGLT2), Aqp1 - Aquaporin 1; TAL (Thick Ascending Limb): Slc12a1 - Na/K/Cl cotransporter 2 (NKCC2), Umod - Uromodulin; DCT (Distal Convoluted Tubule): Slc12a3 - Na/Cl cotransporter (NCC), Pvalb - Parvalbumin; CNT/CD (Connecting Tubule/Collecting Duct): Aqp2 - Aquaporin 2, Scnn1b - Sodium channel epithelial 1 beta subunit (β-ENaC), Scnn1g - Sodium channel epithelial 1 gamma subunit (γ-ENaC); Cldn16 - Claudin 16 and Clcnkb (Cl channel Kb) are localized in both TAL and DCT. (c) Representative Western blot analysis on whole kidney lysates of 12 weeks Kcnj1+/+ and Kcnj1−/− mice. β-actin was used as a loading control. (d) Representative Western blot analysis on kidney membrane fractions of 12 weeks Kcnj1+/+ and Kcnj1−/− mice. Flotillin-1 was used as a loading control, whereas GAPDH was used to test cytosolic contamination. (e) Representative Western blot analysis on kidney cytosolic fractions of 12 weeks Kcnj1+/+ and Kcnj1−/− mice. GAPDH was used as a loading control, whereas Flotillin-1 was used to test membrane contamination. (f) Immunofluorescence analysis for UMOD (green) and NKCC2 (red) in Kcnj1+/+ and Kcnj1−/− serial kidney sections. Scale bars: low magnification - 50 μm, high magnification - 15 μm. Full length blot images can be found in Supplementary Figure S2. less

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Quantifying exosome secretion from single cells reveals a modulatory role for GPCR signaling.

In J Cell Biol on 5 March 2018 by Verweij, F. J., Bebelman, M. P., et al.

Fig.6.F

Fig.6.F showing Western Blotting in a Homo sapiens (Human) sample from the publication: Quantifying exosome secretion from single cells reveals a modulatory role for GPCR signaling.

GPCR activation triggers MVB–PM fusion in HeLa cells via SNAP23-Ser110 phosphorylation. (a) Fusion activity of histamine-stimulated cells nontreated or treated with the Gαq inhibitor UBO-QIC (1 µM). n ≥ 24 cells per condition. (b) Basal fusion activity in cells treated with PKC inhibitors GÖ6976 ... more

GPCR activation triggers MVB–PM fusion in HeLa cells via SNAP23-Ser110 phosphorylation. (a) Fusion activity of histamine-stimulated cells nontreated or treated with the Gαq inhibitor UBO-QIC (1 µM). n ≥ 24 cells per condition. (b) Basal fusion activity in cells treated with PKC inhibitors GÖ6976 (1 µM) or GÖ6983 (1 µM). n ≥ 11 cells per condition. (c) Fusion activity of histamine-stimulated cells nontreated or preincubated with GÖ6983 (1 µM). n ≥ 11 cells per condition. (d) Schematic representation of SNAP23 with SNARE motifs, a membrane-anchoring domain (M), and all phosphosites with the posphosite targeted by histamine stimulation (Ser110) in bold. (e) Fusion activity of histamine-stimulated cells transfected with WT SNAP23, phosphomutant SNAP23-S110A, or phosphomimic SNAP23-S110D. n ≥ 16 cells per condition. (f) Left: fusion activity of CD63-, CD81-, and CD9-pHluorin HeLa cells cotransfected with SNAP23 WT or SNAP23-S110A. n ≥ 16 cells per condition. Western blot on exosomes isolated from SNAP23-WT and SNAP23-S110A HeLa cells labeled for CD63, CD9, CD81, flotillin-1, and syntenin-1. (g) Schematic representation of the histamine-stimulated pathway leading to exosome release as identified by phosphoproteomics and specific inhibitors. Blue-rimmed proteins represent the putative pathway implicated by both experiments. The IP3–Ca2+ pathway is represented in gray as a direct link with MVB–PM fusion is missing. **, P < 0.01; ****, P < 0.0001 using Student’s two-tailed two-sample t test. All t tests were paired except for b and f. Whiskers in the box plots in b and f represent 1.5 times the interquartile distance or the highest or lowest point, whichever is shorter. less

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