Ab133747

Chondrocytes were lysed by radio immunoprecipitation assay (RIPA) lysis buffer with phenylmethylsulfonyl fluoride (PMSF) and a phosphatase inhibitor. The concentration of proteins was measured using a bicinchoninic acid (BCA) protein assay kit (Beyotime, Beyotime Biotechnology, China). The proteins (30 µg) were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and were transferred to polyvinylidenedifluoride (PVDF) membranes. The membranes were blocked in a nonfat dry milk solution for 1 hour. Then, the membranes were incubated overnight at 4°C with anti-AMPK (CST, #2532, 1:1000 dilution), anti-p-AMPK (CST, #2535, 1:1000 dilution), anti-mTOR (CST, #2983, 1:1000 dilution), anti-p-mTOR (CST, #5536, 1:1000 dilution), anti-Beclin-1 (Abcam, ab207612, 1:1000 dilution), anti-ULK1 (Abcam, ab133747, 1:1000 dilution), anti-LC3 II (Abcam, ab48394, 1:1000 dilution), anti-P62 (Abcam, ab56416, 1:2000 dilution), anti-Bcl2 (Abcam, ab194583, 1:2000 dilution) and anti-Bax (Abcam, ab32503, 1:1000 dilution) primary antibodies in dilution buffer. Next, the membranes were incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse IgG (Zhongshan Golden Bridge Biotechnology, 1:5000 dilution) at room temperature for 1 hour. Then, the membranes were developed using the enhanced chemiluminescence substrate LumiGLO (Millipore, Bedford, MA, USA).