Lc3 1

IGF1-R antibody was purchased from Abcam (ab182408, Cambridge, UK). β-Actin antibody was purchased from Thermo Scientific (Rockford, IL, USA). LC3-I, LC3-II, p62, ATG-7, ATG-12, BNIP3, and HIF1-α were purchased from Novus (Novus Biologicals, Centennial, CO, USA). Pro-caspase 3 was purchased from Cell Signaling Technology (Beverly, MA, USA). Pro- and cleaved caspase 9 was purchased from Abcam (Cambridge, UK).

The total protein of cartilage tissues or chondrocytes was prepared by homogenizing the tissues in WIP Tissue and Cell lysis solution (CellChip Beijing Biotechnology Company, Beijing, China) following the manufacturer’s instructions. Protein samples (10 μg each) were loaded on 10% SDS–PAGE and transferred to polyvinylidene fluoride membranes (IPVH00010, Millipore, MA, United States). Membranes were incubated overnight at 4°C with corresponding antibodies. The secondary antibody (IRDye 680RD, LI-COR Biosciences, United States) was diluted at 1:10,000 in Tween-Tris-buffered saline. Blottings were visualized by Odyssey CLx Imaging System (LI-COR Biosciences, United States). GAPDH was used as an endogenous reference. The density of the bands was normalized to that of GAPDH.
Antibodies used for blotting in this study are as follows: BAX (Santa Cruz, sc-7480), Bcl-2 (Abcam, ab196495), Cleaved Caspase-3 (Cell Signaling Tech, #9664), CREB (ProteinTech, 12208-1-AP), GAPDH (Abcam, ab9485), LAMP2 (Cell Signaling Tech, #49067), LC3I (Novus Bio, NB100-2331), LC3II antibody (Novus Bio, NB100-2220), METTL3 (Abcam, ab195352), P62 (Cell Signaling Tech, #5114), and TFEB (Cell Signaling Tech, #4240).