A01388

Monoclonal antibodies against CD63 (mouse; H5C6; 556019; BD), CD81 (mouse; JS-81; 551112; BD), CD9 (mouse; a gift from E. Rubinstein, Institut National de la Santé et de la Recherché Médicale, Villejuif, France), and Alix (3A9; 2171; Cell Signaling Technology) were used at 1:200. Syntenin-1 (rabbit; 610821; BD) and flotillin-1 (rabbit; ab19903; Abcam) mouse antibodies were used at 1:250. Antibodies against SNAP23 (rabbit; 111-202; Synaptic Systems), GFP (rabbit; A01388; Genscript), and β-actin (mouse; sc-47778; Santa Cruz Biotechnology, Inc.) were used at 1:1,000. Secondary anti–mouse Alexa Fluor 594 antibody (goat; A-11032; Invitrogen) was used at 1:500. For Western blotting, cells or exosomes were lysed in a 1% SDS buffer, and equal amounts of protein were loaded onto an SDS-PAGE gel. Only gels for CD63, CD81, and CD9 detection were run under nonreducing conditions.
EGTA-AM (Sigma-Aldrich) was applied at 200 µM for 15 min at 37°C, BAPTA-AM (Sigma-Aldrich) was applied at 20 µM for 15 min at 37°C, and GÖ6983 (Axon Medchem) and GÖ6976 (EMD Millipore) were applied at 1 µM for 2 h at 37°C. GW4869 (Sigma-Aldrich) was applied at 5 µM for 4 h. Histamine (Sigma-Aldrich) was used at 100 µM, and caffeine (Sigma-Aldrich) was used at 20 mM. UBO-QIC (a gift from E. Kostenis, University of Bonn, Bonn, Germany) was used at 1 µM for at least 30 min (preincubation).

Proteins were separated using SDS-PAGE and visualized by staining with Coomassie brilliant blue. Immunoblotting was used to confirm PhaC or Ag85A-ESAT-6-PhaC fusion protein on MBB or the production of GFP. Protein bands separated by SDS-PAGE were transferred to nitrocellulose membranes using the iBlot system (Invitrogen, Carlsbad, CA). Membranes were blocked with 1% skim milk in phosphate-buffered saline with Tween 20 (PBST) for 1 h. Following washing with PBST, primary antibodies were diluted in 1% bovine serum albumin (BSA) and used accordingly: for detection of PhaC, 1:20,000 rabbit polyclonal (GenScript, NJ); ESAT-6, 0.1 μg/ml rabbit polyclonal (Abcam, Cambridge, United Kingdom); and GFP, 0.75 μg/ml rabbit polyclonal (A01388; GenScript, NJ). Following incubation for 1 h, the membrane was washed three times with PBST for 5 min. Secondary antibody anti-rabbit horseradish peroxidase (HRP) at 1:25,000 (Ab6721; Abcam, UK) was diluted in 1% BSA, added, and incubated for 1 h. Following three PBST washes, development was carried out using SuperSignal West Pico chemiluminescent substrate (Thermo Fisher, Waltham, MA).
The BCA protein assay kit (23227; Thermo Scientific, IL) was used according to the manufacturer's instructions to quantify the total protein in the isolated MBB material.

The cells were lysed with cold RIPA buffer supplemented with protease inhibitors, and the extracted proteins were quantified by DC™ Protein Assay Reagent (Bio-Rad Laboratories, Hercules, CA). The extracted protein samples were separated by stain-free SDS-PAGE gels (Bio-Rad Laboratories, 4–15%) and transferred onto Nitrocellulose Membranes (0.45 μm). Primary antibodies include the rabbit polyclonal anti-BVES (1:1000, HPA014788, Sigma-Aldrich, St. Louis, MO), mouse monoclonal anti-Ano5 (1:1000, N421A/85, UC Davis/NIH NeuroMab Facility, Davis, CA), anti-GAPDH (1:4000, MAB374, Cell Signaling Technology, Danvers, MA), anti-GFP antibody (1:1000, A01388, Genscript, Piscataway, NJ), streptavidin-HRP (1:20, DY998, R&D Systems, Minneapolis, MN), myc (1:1000, #2276, Cell Signaling Technology, Danvers, MA), FLAG (1:1000, #F3165, Sigma-Aldrich, St. Louis, MO) and HA (1:1000, #3724, Cell Signaling Technology, Danvers, MA). Secondary HRP-conjugated goat anti-mouse (1:4000), goat anti-rabbit (1:4000) antibodies were obtained from Cell Signaling Technology. The membranes were developed using ECL western blotting substrate (Pierce Biotechnology, Rockford, IL) and images were scanned with the ChemiDoc XRS + system (Bio-Rad Laboratories). Western blots were quantified using Image Lab 6.0.1 software (Bio-Rad Laboratories) according to the manufacturer’s instructions.