Nrf2 antibody

Cells or tissues were added to RIPA lysate (Beyotime, Nanjing, China) and homogenized on a homogenizer (Jingxin, China) adjusted to 60 Hz for 45s. Cells or tissues were centrifuged at 14,000 g for 5 min and the supernatant was removed. A BCA Protein Quantification Kit (Vazyme Biotech, Nanjing, China) was used to detect the protein sample concentration. After electrophoresis using SDS-PAGE gel and membrane transfer, protein-free fast blocking solution (Epizyme, China) was used for blocking. Subsequently, the blocking solution was aspirated and primary antibody was added and incubated overnight at 4°C. After washing three times, the secondary antibody was added and incubated at room temperature for 1 h. The ECL developer (Vazyme Biotech, Nanjing, China) was added and then developed using a fully automated chemiluminescent image analysis system (Tanon, China). The grayscale values of each group of bands were analyzed using ImageJ software. The following antibodies were used: aggrecan antibody, collagen II antibody, Nrf2 antibody, phosphorylated Nrf2 antibody, beta actin antibody (all Affinity Biosciences, China); anti-heme oxygenase 1 antibody (Abcam, Cambridge, UK); and the phospho-MAPK family antibody sampler kit and MAPK family antibody sampler kit (Cell Signaling Technology, Danvers, MA, USA).