Mouse anti cd81

Total protein was extracted from exosomes with procedures as described in detail elsewhere [64 (link)]. Briefly, equal amounts of proteins were added on 4–12% SDS gels for electrophoresis and then transferred to PVDF membranes (ThermoFisher Scientific). The membranes were blocked for 1 h in 5% milk. All the membranes were incubated with primary antibodies at 4 °C overnight. Primary antibodies used in this study were as follows: rabbit-anti-CD9 (Thermo Fisher Scientific, PA5-11559), mouse-anti-CD63 (Thermo Fisher Scientific, 10628D), mouse-anti-CD81 (Thermo Fisher Scientific, MA5-13548), rabbit-anti-GRP94 (Abcam, 13509), mouse-anti-GM130 (Santa Cruz, sc-55591), mouse-anti-GAPDH (HyTest, 5G4MAb6C5). On the next day, the membranes were washed three times for 10 min in TBST (pH = 7.4) and incubated for 1 h with secondary antibodies at room temperature. Secondary antibodies used were anti-Mouse IgG (Fab specific)-Peroxidase antibody produced in goat (Sigma-Aldrich, A3682) and anti-Rabbit IgG (whole molecule)-Peroxidase antibody produced in goat (Sigma-Aldrich, A0545). The results were visualized with chemiluminescence, and the density of the bands was analyzed by ImageJ software.

100 μL of each column fraction (24 in total) was applied to a 0.45 μm nitrocellulose membrane (Bio-Rad), prewetted in Tris-buffered saline (TBS), in a BioDot apparatus (Bio-Rad). Membranes were blocked in 5% (w/v) nonfat dry milk (Bio-Rad) in TBS with 0.1% (v/v) Tween-20 (Sigma) for 1 hour at room temperature, washed three times 10 minutes in TBS-T and incubated overnight at 4 °C in one of three primary antibodies: mouse-anti-CD9, (Thermo, 10626D), mouse-anti-CD63, (Thermo, 10628D), or mouse-anti-CD81, (Thermo, 10630D) all diluted 1 : 1000 in 5% (w/v) bovine serum albumin (BSA, Sigma) in TBS-T. The next day, membranes were washed three times 10 minutes in TBS-T and incubated for 1 hour at room temperature with LiCor-dye 800 CW-conjugated goat-anti-mouse (LiCor Biosciences) secondary antibody diluted 1 : 10 000 in TBS-T. After another three 10 minute washes in TBS-T, membranes were imaged on a LiCor Odyssey near-infrared imager (LiCor). Blots were quantified using the LiCor ImageStudio software.