Radioimmunoprecipitation assay buffer lysis buffer

For western blot analysis, the total protein was extracted by radioimmunoprecipitation assay buffer lysis buffer (Beyotime, Shanghai, China) with the concentration measured using a Pierce™ bicinchoninic acid Protein Assay Kit (ThermoFisher). Thereafter, proteins were separated with 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, followed by electrophoretic transfer into polyvinylidene difluoride membranes. After being blocked using non-fat milk, the membranes were incubated at 4°C with anti-rabbit Bax (1:1000 dilution, ab32503, Abcam), Bcl-2 (1:1000 dilution, ab32124, Abcam), FOXO1 (1:1000 dilution, ab39670, Abcam) or GAPDH (1:5000 dilution, ab8245, Abcam) antibodies overnight, rinsed with Tris buffered saline containing Tween 20 and further incubated with secondary anti-rabbit antibody (1:5000 dilution, A0208, Beyotime) at room temperature for 1 h. Finally, the blots were observed by using the enhanced chemiluminescence solution (ThermoFisher) with the signals analyzed by Image Lab™ Software 5.2 (Bio-Rad, Hercules, CA, USA).

The chondrocytes were lysed with pre-chilled radioimmunoprecipitation assay buffer lysis buffer (Beyotime), and then quantified using a bicinchoninic acid protein detection kit (Solarbio, Beijing, China). The protein samples (50 µg) were isolated by 10% SDS-PAGE, and the nitrocellulose membranes were transfected (Millipore, New York, NY, USA). Next, the membranes were incubated overnight with primary antibodies at 4 ℃, and then with secondary antibodies for 2 hours at room temperature. The membranes were developed by chemiluminescence signal with an enhanced chemiluminescence imaging kit (Thermo Fisher Scientific, Waltham, MA, USA, Cat. #32209).