Invitrogen ibright fl1000 imaging system

The chondrocytes were lysed by radioimmunoprecipitation assay buffer with protease inhibitor cocktail (Roche, Indianapolis, IN, USA) for protein extraction, and protein concentration was determined using the bicinchoninic acid (BCA) assay (23227, Pierce™ BCA protein assay kit, Thermo Fisher Scientific, Waltham MA, USA). Twenty μg of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA), and incubated with anti-phospho-Nrf2 (ab180844, Abcam, Cambridge, UK), anti-Nrf2 (ab62352, Abcam, Cambridge, UK), anti-nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB, 10745-1-AP, Proteintech, Rosemont, IL, USA), anti-p50 (14220-1-AP, Proteintech, Rosemont, IL, USA), anti-cyclooxygenase-2 (COX-2, 66351-1-lg, Proteintech, Rosemont, IL, USA), and anti-β-actin (MA5-15739, Thermo Fisher Scientific, Waltham MA, USA) antibodies at 4 °C overnight. Protein expression was detected with a chemiluminescence reagent (SuperSignal West Pico PLUS Chemiluminescent Substrate, Thermo Fisher Scientific, Waltham MA, USA) and imaged using a Western blot imaging/gel documentation system (Invitrogen iBright FL1000 Imaging System, Thermo Fisher Scientific, Waltham MA, USA). The band of each targeting protein was quantified and expressed as relative densitometry.