Cleaved cas 3

Total protein was extracted using radioimmunoprecipitation assay (RIPA) buffer (Solarbio, Beijing, China). Equal protein for each sample was separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then electrophoretically transferred to polyvinylidene fluoride membranes (GE Healthcare, Piscataway, NJ, USA). Subsequently, membranes were blocked with 5% skim milk solution at room temperature for 1 h. Protein expression was detected through incubation of the membranes with indicated primary antibody at 4°C overnight. After being washed three times for 10 min each, membranes were incubated with Goat polyclonal Secondary Antibody to Rabbit IgG-H&L (ab1500771; 1:2,000 dilution; Abcam, Cambridge, MA, USA) for 1 h at room temperature. ECL Western Blotting Detection Kit (Solarbio) and Image J software (National Institutes of Health, Bethesda, MD, USA) were used to visualize and quantify protein expression, correspondingly. The primary antibodies used were proliferating cell nuclear antigen (PCNA; ab92552; 1:1,000 dilution; Abcam), Cleaved-caspase-3 (Cleaved-cas 3; ab2302; 1:1,000 dilution; Abcam), matrix metalloproteinase 2 (MMP-2; ab97779; 1:1,000 dilution; Abcam), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; ab181602; 1:3,000 dilution; Abcam).

For total protein preparation, chondrocytes were treated with pre-cold RIPA buffer (Beyotime, Nantong, China), followed by quantification using bicinchoninic acid (BCA) Protein Assay Kit (Solarbio, Beijing, China). For immunoblotting, the protein samples (50 μg) were separated by 10% SDS-PAGE and electrophoretically transfected to nitrocellulose membranes (Millipore, New York, NY, USA). And then, the membranes were subjected to hybridize with primary antibodies: cleaved cas-3 (1:1000, Abcam, Cambridge, MA, USA), Bcl-2 (1:1000, Abcam, Cambridge, MA, USA), MMP-13 (1:1000, Abcam, Cambridge, MA, USA), COL2A1 (1:1000, Abcam, Cambridge, MA, USA), IL-6 (1:1000, Abcam, Cambridge, MA, USA), IL-8 (1:1000, Abcam, Cambridge, MA, USA), and GAPDH (1:1000, Abcam, Cambridge, MA, USA) at 4°C overnight. After incubation with secondary antibody (1:10,000, Abcam, Cambridge, MA, USA) for 2 h, the detection of protein bands was performed using ECL detection system (GE Healthcare, Piscataway, NJ, USA).