Adamts5 ab41037

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).