Western Blot Analysis of Mitochondrial Proteins

Mitochondrial, cytoplasmic and total proteins were extracted, and the corresponding kit (Beyotime) was used to detect the content. Thereafter, 25 µg of protein was subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis. The protein was transferred to a polyvinylidene fluoride film (Millipore, Billerica, Massachusetts, USA) using a semidry method. The polyvinylidene fluoride film was soaked in TBST containing 5% skimmed milk powder and sealed with a shaker for 2 h at room temperature. The blots were incubated overnight, with the primary antibodies diluted from 1:500 to 1:1000. The antibodies were used against the proteins listed below: Parkin (ab77924), PINK1 (ab23707), Bax (ab32503), Bcl-2 (ab32124), Cyt-c (ab110325), Collagen II (ab34712), Adamts5(ab41037) (Abcam, Cambridge, UK); P62 (#5114), LC3 (#2775), SIRT3 (#2627S), GAPDH (#5174), Caspase-3 (#9662) and Cleaved-Caspase-3 (#9664) (Cell Signaling Technology; Danvers, Massachusetts, USA); VDAC1 (sc-32063) (Santa Cruz Biotechnology; Dallas, Texas, USA); Aggrecan (13880-1-AP) and MMP3 (17873-1-AP) (Wuhan Sanying, Wuhan, China). After rinsing the film, the proper secondary antibody was incubated through the blot for 1 h at 25 °C. The film’s gray values were analyzed after darkroom exposure using Image J software v1.46 (NIH, Bethesda, MD, USA).