Western Blot Analysis of Chondrocytes

Western blot analysis was performed as described previously^{10 (link)}. Briefly, the total cellular protein was isolated from human and mouse chondrocytes and SW1353 cells using radioimmunoprecipitation assay lysis buffer (Beyotime Biotechnology) containing protease inhibitors (1:100, Abcam, Cambridge, UK). Electrophoresis was started at 80 V for 20 min and then continued using 120 V for 1 h, followed by 250 mA transfer for 1.5 h. After blocking for 15 min with protein-free rapid blocking buffer (PS108, Epizyme, Shanghai, China), the membranes were incubated overnight at 4 °C with primary antibodies (Supplementary Table 2). Following incubation, the membranes were treated with the corresponding horseradish peroxidase-conjugated secondary antibodies (1:3000, Cell Signaling Technology, Danvers, MA, USA) at 20–25 °C for 1 h. The protein bands were detected using a ChemiDoc Touch (Bio-Rad Laboratories, Hercules, CA, USA) and analyzed using Image Lab^TM (Bio-Rad Laboratories). The intensity of bands was compared using ImageJ software (NIH, Bethesda, MD, USA).