Chondrocyte Protein Extraction and Analysis

RIPA Lysis Buffer (Thermo Fisher Scientific) was used for total protein extraction from chondrocytes, and the extracted proteins were separated using SDS-PAGE and transferred to PVDF membranes, which were incubated in 5% milk for 2 h at room temperature to block nonspecific binding. After washing thrice with PBS buffer, the membranes were incubated (overnight at 4°C) with primary antibodies against autophagy-related gene (ATG) 7, LC3-II/LC3-I, PI3K, p-AKT1, AKT1, p-mTOR, mTOR, p70S6K, p-p70S6K, or GAPDH; all primary antibodies were from Cell Signaling Technology (CST; Boston, MA, USA) and were used at 1:1000 dilution. Next, the membranes were incubated with anti-rabbit IgG antibody (1:1000, CST) for 2 h at room temperature, and then the immunoreactive proteins on the membranes were detected using an imaging system (Bio-Rad, Hercules, CA, USA) [23 (link)].