Journal: Brain

Article Title: Reduced oligodendrocyte exosome secretion in multiple system atrophy involves SNARE dysfunction

doi: 10.1093/brain/awaa110

Figure Lengend Snippet: α-Syn-syntaxin 4 interaction regulates SNARE complex function and exosome secretion. (A) The binding of syntaxin 4 with α-syn. Lysates of human oligodendrocytic MO3.13 cells were immunoprecipitated with an anti-α-syn antibody and subjected to western blots with antibodies against syntaxin 4 or α-syn. The interaction between syntaxin 4 and α-syn was demonstrated by the co-immunoprecipitation of syntaxin 4. (B) Reciprocal co-immunoprecipitation using an anti-syntaxin 4 antibody for immunoprecipitation confirmed the interaction. (C) The co-localization of α-syn and syntaxin 4 was also observed in MO3.13 cells using confocal microscopy. Cells were stained with antibodies against α-syn (green) and syntaxin 4 (red) as well as with DAPI (blue). Scale bar = 10 μm. Arrows indicate the co-localization of α-syn and syntaxin 4. (D) MO3.13 cells were transfected with the α-syn overexpressing (pCMV-tag2b-SNCA) plasmid or the empty vector, and the interaction between syntaxin 4 and VAMP2 was reduced when overexpressing α-syn, as shown by the decrease of VAMP2 co-immunoprecipitated with syntaxin 4, while the total amounts of syntaxin 4 and VAMP2 were not changed. Inputs (cell lysates) are shown with images acquired with shorter exposures from the same gel. (E and F) MO3.13 cells were treated with increased concentrations of recombinant α-syn proteins (0, 100, 200, and 500 pg/ml), and the amount of VAMP2 interacted (co-immunoprecipitated) with syntaxin 4 was quantified using western blots. The syntaxin 4-VAMP2 binding was negatively associated with recombinant α-syn concentrations. Error bars represent the standard deviation from the mean; *P < 0.05. IP = immunoprecipitation.

Article Snippet: For immunoprecipitation, 500 μg (total protein) of cell lysates were incubated with anti-α-syn (Cat# ab138501, Abcam, 1:250), anti-syntaxin 4 (Cat# 14988-1-AP, Proteintech, 1:40), or anti-VAMP2 (Cat# ab181869, Abcam, 1:200) overnight at 4°C.

Techniques: Binding Assay, Immunoprecipitation, Western Blot, Confocal Microscopy, Staining, Transfection, Plasmid Preparation, Recombinant, Standard Deviation

Journal:

Article Title: The Platelet Actin Cytoskeleton Associates with SNAREs and Participates in ?-Granule Secretion †

doi: 10.1021/bi100541t

Figure Lengend Snippet: A, increased expression of P-selectin is observed following exposure of the α–granule enriched membrane fraction to 5 mM ATP plus 4 mg/ml platelet cytosol. B, P-selectin expression was monitored following incubation of the α–granule enriched membrane fraction with buffer alone (NA), with 5 mM ATP (ATP), with 4 mg/ml platelet cytosol (cytosol), or with ATP plus cytosol (ATP+cytosol). Data represent the mean ± S.D. of 3-4 experiments. Insert demonstrates release of β-thromboglobulin from α–granule-enriched fraction following exposure to 4 mg/ml platelet cytosol and 5 mM ATP. C, P-selectin expression was monitored following incubation of α–granule-enriched membrane fraction with buffer alone (NA), or in the presence of 5 mM ATP plus 4 mg/ml cytosol following addition of 25 μg/ml of either non-immune IgG (NI), anti-syntaxin 4 antibody (anti-synt-4) or anti-P-selectin cytoplasmic tail antibody (anti-PSCT). Following a 20 minute incubation, the sample was exposed to 5 mM ATP and 4 mg/ml platelet cytosol and analyzed for P-selectin expression by flow cytometry. Data represent the mean ± S.D. of 3 experiments.

Article Snippet: Mouse anti-syntaxin-4 antibody was obtained from BD Biosciences (San Jose, CA).

Techniques: Expressing, Incubation, Flow Cytometry

Journal:

Article Title: The Platelet Actin Cytoskeleton Associates with SNAREs and Participates in ?-Granule Secretion †

doi: 10.1021/bi100541t

Figure Lengend Snippet: A, platelets were lysed in 1.5 ml of Triton X-100 lysis buffer at 4°C. Samples were subject to centrifugation at 15,000g to isolate the platelet cytoskeletal actin network. The supernatant was removed and the pellet washed. Samples of the pellet (Pellet, 30% of total) and the supernatant (Sup, 1% of total) were solubilized in sample buffer and proteins within the samples separated by SDS-PAGE. SNAP-23, syntaxin-2, syntaxin-4, and VAMP-8 within the samples were detected by immunoblot analysis. B, platelets were incubated in the presence (+) or absence (−) of SFLLRN and lysed in Triton X-100 lysis buffer containing either EGTA. Lysates were then spun at 15,000g. Proteins in the pellet were separated by SDS-PAGE and analyzed for SNAP-23, syntaxin-2, syntaxin-4, and VAMP-8 by immunoblot analysis.

Article Snippet: Mouse anti-syntaxin-4 antibody was obtained from BD Biosciences (San Jose, CA).

Techniques: Lysis, Centrifugation, SDS Page, Western Blot, Incubation

Journal:

Article Title: The Platelet Actin Cytoskeleton Associates with SNAREs and Participates in ?-Granule Secretion †

doi: 10.1021/bi100541t

Figure Lengend Snippet: A, proteins were incubated alone (GST alone; synt-4-GST alone) or in the presence of polymerized platelet actin (GST, actin; synt-4-GST, actin). Samples were pelleted by centrifugation. Proteins within supernatants and pellets were separated by SDS-PAGE and visualized by Coomassie blue staining. B, recombinant GST alone, syntaxin-4-GST, α-actinin, or SNAP-23-GST were incubated in the presence (black bars) or absence (white bars) of polymerized platelet actin. Samples were pelleted and proteins within the supernatants and pellets separated by SDS-PAGE. Bands corresponding to each of the proteins were quantified by densitometry. Data represent the ratio of band density in the pellet compared to that in the supernatant and are representative of 3-5 experiments.

Article Snippet: Mouse anti-syntaxin-4 antibody was obtained from BD Biosciences (San Jose, CA).

Techniques: Incubation, Centrifugation, SDS Page, Staining, Recombinant