Vdac1 b 6

Commercially available antibodies used for this study include mouse monoclonal FLAG M2 antibody (1:5000) (Sigma); rabbit polyclonal V5 antibody (1:5000) (Thermo Fisher); mouse monoclonal V5 (1:5000), β-actin (1:5000), and TBK1 antibody (1:1000) (Abcam); Phospho-TBK1 (Ser172) (1:1000), Phospho-IRF3 (Ser396) (1:1000), Ku80 (1:1000), cGAS (D1D3G) (1:1000), and STING (D2P2F) antibody (1:1000) (Cell Signaling); GAPDH (6C5) (1:1000), VDAC1 (B-6) (1:1000), and IRF3 antibody (FL-425) (1:1000) (Santa Cruz); DNA-PKcs (1:1000) and Ku70 antibody (1:1000) (NeoMarkers); and phospho-Histone H2A.X (Ser139) antibody (1:1000) (Millipore).
Rabbit polyclonal anti-cGAS pT68 and pS213 antibodies were generated by GenScript Company (NJ, USA) and used at 1:500 dilution. Rabbit polyclonal anti-MAVS antibody was generated in the laboratory and used at 1:1000 dilution^{48 (link)}.
Nu7441 (Selleck Chemical), Nu7026 (MedChemExpress), Herring testis (HT)-DNA (Sigma-Aldrich), poly I:C (InvivoGen), 2’,3’-cGAMP (InvivoGen), streptavidin agarose (Thermo Fisher), Ni-NTA His-Bind Resin (Novagen), and Malachite Green Phosphate Detection Kit (Cell Signaling) were used according to the manufacturer’s protocol. Biotin-labeled ISD-45 DNA was ordered from IDT. [α-P32]-ATP was ordered from Perkin Elmer. Lipofectamine 2000 was purchased from Invitrogen.

EV sample size distributions were measured using a NanoSight NS300 (Malvern Panalytical) equipped with a 532 nm laser, where EV samples were analyzed for 30 s, in triplicate, using NanoSight particle tracking software (screen gain and detection threshold set as 1.0 and 2, respectively). The morphology of EV samples negatively stained with osmium tetroxide were analyzed using a 2010F transmission electron microscope (JEOL USA). Western blots analyses were performed according to standard protocols using 20 µg (~ 8 μl) of cell and EV protein lysates that were probed with antibodies to TSG101 (4A10; Santa Cruz Biotechnology), VDAC1 (B-6; Santa Cruz Biotechnology), and EpCAM (VU-1D; Invitrogen), and a horseradish peroxidase (HRP)-coupled secondary antibody (RMG07; Abcam). Chemiluminescent signal from these Western blots was visualized using an ImageQuant™ LAS 4000 imaging system (GE Healthcare Life Sciences).