Anti atg12

Cells were lysed in RIPA lysis buffer, and cell lysates were harvested. Protein (20 µg) was loaded for electrophoresis and subsequently transferred onto PVDF membranes (GE healthcare, Chicago, IL, USA). Membranes were blocked in 8% non-fat milk solution and incubated with rabbit anti-LC3 (1:2000, Abcam, Cambridge, UK), anti-Beclin1 (1:1000, Abcam), anti-p62 (1:1000, Abcam), anti-ATG12 (1:2000, Abcam) and anti-GAPDH (1:8000, Abcam). Subsequently, membranes were incubated with an HRP-conjugated goat anti-rabbit IgG antibody (Abcam) for 1 h. The bands were visualized using the ECL substrate (Beyotime). Image J software was used to analyze band intensity.

The human articular chondrocyte cell line HC-a (Sciencell, Carlsbad, CA, USA) was maintained in DMEM supplemented with 15% fetal bovine serum, plus antibiotics. SW1353 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and were maintained in L-15 medium (Gibco, Grand Island, NY, USA). OUMS-27 cells, HCS-2/8 cells and JJ012 cells were kindly gifted from Dr. J Block (Rush Medical College, Chicago, IL, USA) and were cultured in Dulbecco's modified Eagle's medium (Hyclone, Logan, UT, USA) supplemented with 10% fetal calf serum (Gibco) at 37 °C in a humidified atmosphere with 5% CO₂.
Rapamycin and 3MA were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The following antibodies were used in the experiments: anti-p-Stat3, anti-Stat3, anti-cyclin D1, anti-bcl-2, anti-Bax, anti-LC3, anti-p62 and anti-GAPDH were from Cell Signaling Technology (Beverly, MA, USA). Anti-c-Myc and anti-Atg12 was from Abcam (Cambridge, MA, USA).