Exoab cd63a 1

sEV samples were lysed in ice-cold RIPA buffer (Beyotime, China) on ice for 15 min and centrifuged at 13,000 g for 10 min. The protein concentration in the supernatant was determined via the BCA assay (Pierce, NCI225CH). Thirty micrograms of total protein was separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted onto a PVDF membrane (Millipore, USA). The membrane was blocked with 5% nonfat milk PBST for 1 h at RT and then incubated with primary antibodies against CD63 (rabbit polyclonal, System Biosciences, EXOAB-CD63A-1), CD81 (rabbit recombinant monoclonal, Abcam, ab109201), CD9 (rabbit polyclonal, System Biosciences, EXOAB-CD9A-1), calnexin (rabbit polyclonal, Proteintech, 10427-2-Ap) and albumin (rabbit polyclonal, Proteintech, 16475-1-AP) overnight. After incubation with a goat anti-rabbit HRP secondary antibody (Jackson Immunoresearch, West Grove, PA, 111-035-003) for 1 h at RT, protein bands were visualized using an enhanced chemiluminescent (ECL) substrate (Tanon, Shanghai, China, 180–501).

EVs were processed for immunoblotting using established protocols. Samples were homogenized and resuspended in radioimmunoprecipitation assay (RIPA) buffer with protease inhibitor cocktail (Sigma), rotated for 2 h at 4 °C, and pelleted at 5000 × g for 10 min. Protein quantification was done using Bradford assay (BioRad). For immunoblotting, 20 μg of protein was loaded per lane for gel electrophoresis onto a NuPAGE 4–12% Bis-Tris gel (Life Technologies). After running, gels were cut and the same molecular weight sections for all samples were transferred together to enable multiple probing and to control for transfer conditions. After transfer of proteins to a nitrocellulose membrane (Life Technologies), membranes were blocked with Odyssey blocking buffer (Li-Cor) and probed with rabbit anti-CD63 (1:1000; Systems Biosciences EXOAB-CD63A-1), rabbit anti-Calnexin (1:1000; Abcam ab22595), and/or rabbit anti-Lamp1 (1:1000; Abcam ab24170), followed by incubation in IRDye800-conjugated donkey anti-rabbit secondary (1:20,000; Li-Cor).