Antibody against cd63

Isolated exosome pellet was lysed in RIPA (Radio‐Immunoprecipitation Assay) buffer supplemented with complete protease inhibitor cocktail tablets (Roche, Basel, Swiss). Cultured HUVECs were harvested and lysed in RIPA buffer supplemented with complete protease inhibitor cocktail tablets. Cell debris was removed by centrifugation at 13,400 g for 20 min. Then the lysates of exosomes or cells were separated by SDS‐PAGE gels, transferred to PVDF membranes (Bio‐Rad, California, USA), and incubated with the relevant antibodies as indicated. Quantity One software was used to calculate the abundance of protein. Antibody against CD63 was purchased from Santa Cruz Biotechnology (California, USA), p‐p105 and Alix were purchased from Cell Signaling Technology (Massachusetts, USA) and VCAM‐1, TSG101 and Calnexin were purchased from Abcam (Cambridge, UK).

Exosomes were lysed using the Total Exosome RNA and Protein Isolation Kit (Invitrogen, U.S.A.) in accordance with the manufacturer’s instructions, and exosomal proteins were extracted for Western blot. Proteins were electrophoresed through SDS/PAGE gel and transferred to PVDF membranes. CD63 protein level was quantitated using antibody against CD63 (Santa Cruz Biotechnology, U.S.A.). β-actin (Sigma, Germany) was used to normalize for protein loading. Bound proteins were visualized by the ECL Plus Kit (Millipore, U.S.A.) with Image Lab Software (Bio-Rad, U.S.A.).

The exosome pallet was lysed in RIPA buffer supplemented with complete protease inhibitor cocktail tablets (Roche). Lysates were separated by SDS-PAGE gels, transferred to PVDF membranes (Bio-Rad), and incubated with the relevant antibodies as indicated. Antibody against CD63 was purchased from Santa Cruz Biotechnology, Alix was purchased from Cell Signaling Technology and TSG101, Calnexin and CCR7 were purchased from Abcam.