Second antibody

Western Blot was used to identify the expressions of CD9 and CD63 on the surface of exosomes and the expressions of Bax, Bid, Bim, Bcl-2, cleaved caspase-3, and cleaved caspase-9 in NRK cells. Total proteins were obtained using lysis buffer, and then quantitated by bicinchoninic acid kit (Beyotime, Shanghai, China). Following sample separating and transferring into PVDF membranes, membranes were immerged in 5% nonfat milk. Next, primary antibodies of CD9, CD6, Bax, Bid, Bim, Bcl-2, cleaved caspase-3, cleaved caspase-9 (1: 800, Abcam), and β-actin (1: 1000, Beyotime), respectively, were used for immunoblotting of the membranes overnight at 4°C. After the incubation with second antibody (1:5000, Jackson, USA), the protein levels were detected by enhanced chemiluminescence (ECL, Millipore, USA).

To determine the protein levels in breast cancer cells, total proteins were extracted and Western blot analyses were performed as described previously16 (link). Electrophoresis was carried out using a 12% SDS-polyacrylamide gel (50 μg protein per lane). Separated proteins were transferred onto PVDF membranes, treated with 1% BSA/0.02% NaNO₃ to block the non-specific IgGs, incubated for 1 h with specific primary antibody (0.2 μg/mL), and then incubated with second antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) linked to horse radish peroxidase (1:10,000) for 1 h. Subsequently, the blot was developed using the ECL (enhanced chemiluminescence) system (GE, Healthcare, NJ). The intensity of each band was quantified by densitometry analysis using Image Pro-Plus 4.5 Software.

Rat chondrocytes were treated according to the above design. The collected cells were lysed by RIPA lysis buffer (Gibco), and then proteins were extracted by commercial kit (Pierce, Rockford, IL, USA). Next, after resolved on PAGE gel, protein sample was transferred to PVDF. Afterwards, the membrane was blocked, and reacted with integrin β1, pro-caspase-3, pro-caspase-9, cleaved caspase-3, cleaved caspase-9 or β-actin primary antibody (1:300, Santa Cruz) overnight at 4°C. After the incubation with second antibody (1:5000, Jackson, USA), the protein levels were detected by enhanced chemiluminescence (ECL, Millipore, USA).