Anti collagen 2

Total protein was extracted, and the protein concentration was quantified using a BCA protein assay kit (Beyotime Biotechnology, Jiangsu, China). A total of 20 μg of protein from each sample was used for Western blotting. The samples were separated by SDS-PAGE(10%) at 200 V for 50 min. After transferring the proteins onto polyvinylidene fluoride (PVDF) membranes, the blotting was performed at 300 mA for 45 min. After blocking with 5% (w/v) dry milk in TBS for 1 h at room temperature. Membranes were incubated with the primary antibodies. The primary antibodies used in this study included monoclonal anti-Collagen II (1:1000), Aggrecan (1:500) (Cell Signaling Technology, Beverly, MA), Fos (1:500), p38 (1:500), JNK (1:500) and their phosphorylated antibodies (1:500, Santa Cruz, CA, USA) and anti-GAPDH polyclonal antibody (1:2000 Santa Cruz, CA, USA) at 4 °C overnight. Then the membranes were incubated with HRP-conjugated anti-rabbit or anti-mouse antibody (1:10000, Cell Signaling Technology, Beverly, MA) for 2 h at room temperature. Finally, the blots were developed with an enhanced chemiluminescence kit (Millipore, Billerica, MA, USA), and the bands were quantified densitometrically using a Bio-Rad imaging system (Hercules, CA). GAPDH was used as the loading control.