mouse anti vamp4  (Proteintech)

Journal: bioRxiv

Article Title: Apoptotic Extracellular Vesicles (ApoEVs) Safeguard Liver Homeostasis and Regeneration via Assembling an ApoEV-Golgi Organelle

doi: 10.1101/2021.02.24.432630

Figure Lengend Snippet: (A) Representative immunofluorescent images show apoEV (labeled with CellMask™ Deep Red, red) surface expression of vesicle-localized-soluble N -ethylmaleimide-sensitive factor attachment protein receptors (v-SNAREs, green) VAMP3, V VAMP4 and Golgi SNARE of 15 kDa (GS15). Flow cytometric analysis was performed to determine percentages of positively stained apoEVs. Scale bars = 25 μm. (B) Western blot analysis show expression of VAMPs in apoEVs compared to their source MSCs. (C) Western blot analysis show expression of VAMPs in apoEVs. Source MSCs were transfected with small interferon RNAs (siRNAs) as a negative control (siCtrl) for VAMP3 (si Vamp3 ), VAMP4 (si Vamp4 ), and both VAMP3 and VAMP4 (si Vamp3/4 ). Flow cytometric analysis was performed to determine percentages of positively stained apoEVs after knockdown of VAMPs. (D) Representative images show cultured Fas mutant ( Fas mut ) tetraploid hepatocytes (4N-HCs) with 130 kDa Golgi matrix protein (GM130) immunofluorescent staining (green), PKH26-labeled apoEVs (red), and DAPI counterstain (blue). White dashed lines depict cell borders of cultured HCs. Imaging analysis was performed to quantify apoEV uptake and percentages of Golgi-contacted apoEVs. Scale bars = 10 μm. N = 4 per group. (E) Representative fluorescent images show cultured Fas mut 4N-HCs with ac-α-tubulin immunostaining (white), N -acetylgalactosaminyltransferase 2 (GALNT2)-GFP-labeled Golgi (green), PKH26-labeled apoEVs (red), and DAPI counterstain (blue). Imaging analysis was performed to quantify HC percentages with Golgi fragmentation and acetylated microtubules (MTs) fluorescence intensity. Scale bars = 5 μm. N = 4 per group. (F) Western blot analysis showed human VAMP3 (hVAMP3) expression in isolated HC Golgi. ApoEVs were derived from human MSCs (hApoEVs). GM130 was used as an internal control. (G) Western blot analysis showed VAMP3 expression in apoEVs without or with treatment by 250 ng/ml Tetanus toxin (TeNT). (H) Representative Golgin84 immunofluorescent staining (green) images of liver Golgi counterstained with DAPI (blue). WT, wild type. Imaging analyses were performed to quantify HC percentages with Golgi fragmentation. Scale bars = 5 μm. N = 4 per group. (I) Golgi apparatus was isolated from the liver and Golgi protein mass was determined using the BCA method. N = 4 per group. (J) Representative liver fluorescent images showed HCs with different nuclei (blue, DAPI for DNA) and their cell borders (green, phalloidin for F-actin). # indicates binucleated HCs. Scale bars = 25 μm. N = 4 per group. (K) After PI staining, percentages of binucleated HCs were quantified. Diploid HCs (2N-HCs) were analyzed using flow cytometry. N = 4 per group. (L) Hematoxylin and eosin (H&E) staining of liver tissues in periportal vein (PV) areas. Hepatic injury scores were examined based on pathological parameters. Scale bars = 50 μm. N = 4 per group. (M) Serum alanine aminotransferase (ALT) levels were determined. N = 4 per group. (N) Graphical summary illustrating that apoEVs use VAMP3 to assemble with Golgi for HC and liver regulation. Data represent mean ± standard deviation. *, P < 0.05; **, P < 0.01; ***, P < 0.0001; NS, not significant, P > 0.05. Statistical analyses were performed by Student’s t test for two-group analysis or one-way analysis of variance followed by the Newman-Keuls post hoc tests for multiple group comparisons.

Article Snippet: For IF staining of surface markers of apoEVs, after isolation, apoEVs were stained with rabbit anti-human/mouse VAMP3 primary antibody (13640, Cell Signaling Technology, USA), rabbit anti-mouse VAMP4 primary antibody (PA1-768, Invitrogen, USA), mouse anti-mouse GS15 primary antibody (610960, BD Transduction Laboratories, UK), rabbit anti-mouse C1q primary antibody (ab71940, Abcam, UK), or mouse anti-mouse TSP1 primary antibody (sc-59887, Santa Cruz Biotechnology, USA) at 4°C for 1 h at a concentration of 1:100 in PBS.

Techniques: Labeling, Expressing, Staining, Western Blot, Transfection, Negative Control, Cell Culture, Mutagenesis, Imaging, Immunostaining, Fluorescence, Isolation, Derivative Assay, Flow Cytometry, Standard Deviation