Western blotting

Protein was isolated by incubating the isolated EVs in RIPA buffer (Sigma-Aldrich) for 15 min at room temperature, followed by sonication. Approximately 20 µg of protein was loaded per well into SDS-PAGE gel, and proteins were separated by SDS polyacrylamide gel electrophoresis using a mini gel tank electrophoresis system (Invitrogen Life Technologies) and then transferred to Immobilon-FL polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membrane containing the transferred protein was probed with primary mouse polyclonal anti-TSG-101 (1:1000 SC-7964, Santa Cruz Biotechnology, USA) primary goat anti-CD63 (1:3000, STJ140029, St. Johns Laboratory, London, United Kingdom), primary CD9 Monoclonal Antibody (IVA50) (1:6000, MA1-19301, Invitrogen) and primary Anti-Calnexin (CNX) Monoclonal Antibody (CAA280Hu22, Cloud Clone Corp, USA)⁶⁹. Membranes were washed in Tris-buffered saline (TBS- pH 7.6) and incubated for 2 h in TBST (TBS with Tween-20) containing 5% BSA along with horseradish peroxidase-conjugated anti-mouse secondary antibody (1:10000, SC-516102, Santa Cruz Biotechnology), anti-goat secondary antibody (1:20000, {"type":"entrez-protein","attrs":{"text":"STJ99512","term_id":"1439666271","term_text":"STJ99512"}}STJ99512, St. Johns laboratory), anti-mouse secondary antibody ( for CD9-1:30000, for Calnexin- 1:10000, A9044, Sigma) respectively. The Substrate Pierce ECL Western Blotting kit (32106) was utilized in the next step for chemiluminescence, and subsequently, the membranes were exposed to X-ray film for 1–5 min before visualization.