Ecl chemiluminescence reagent

Western blot analysis was conducted as described previously (29 (link)). Rabbit knee joints' cartilage tissue samples from each group were dissected, homogenized, and the tissue homogenates were immediately centrifuged twice at 12,000 × g for 15 min at 4°C to isolate supernatant for subsequent western blot analysis. The total protein extracted was quantified using the bicinchoninic acid protein assay kit (BCA kit, Thermofisher, USA). Equal proportions of proteins were transferred to polyvinylidene fluoride (PVDF) membranes following separation by 10% SDS-PAGE (Beyotime,China). The membrane was blocked in 5% bovine serum albumin (BSA, Sigma, USA) followed by incubation with primary antibodies against Nrf2, HO-1, MMP-3, and GAPDH (Affinity, China) overnight at 4°C. The next day, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (goat anti-rabbit/anti-mouse IgG, Affinity, China) for 1 h at room temperature. Western blotting analysis was performed by visualization of bands using the ECL chemiluminescence reagent (Vazyme, China). Using the Tannon automated gel image analysis technology (Shanghai, China), protein bands were detected. To examine the relative expressions of proteins, the Image J software was utilized.

The concentration of the extracted protein was measured with the bicinchoninic acid (BCA) reagent kit (Beyotime Biotechnology Co., Shanghai, China). Equal amounts (30 μg) of total protein was mixed with SDS loading buffer and boiled for 10 min at 100°C and resolved on 10% SDS-PAGE for 30 min at 80V and then at 120V for 1h. Then, the proteins were wet transferred onto a PVDF membrane for 90 min at 100V. The membrane was blocked in 5% BSA (Beyotime Biotechnology Co., Shanghai, China) at room temperature for 1h, and then probed with primary antibodies against GSTM1 (ab113432, 1: 1000), 78 kDa glucose-regulated protein (GRP78) (ab173613, 1: 100), p-PERK (CST, #3179, 1: 1000), p-IRE1α (NB100-2323, 1: 1000) and C/EBP homology protein (CHOP) (ab10444, 1: 250), GAPDH (CST, #2118, 1: 1000) and superoxide dismutase (SOD) (ab13533, 1: 5000) overnight at 4°C. The membranes were then washed by TBST thrice for 5 mins and incubated with the corresponding secondary antibodies for 1h. The blots were then washed in TBST thrice for 5 min and developed with ECL chemiluminescence reagent (Nanjing Vazyme Biotech Co. Ltd, Nanjing, China) and imaged by the Bio-Rad Gel Dol EZ imager (GEL DOC EZ IMAGER, Bio-Rad, CA, USA). GAPDH was used as internal reference and the relative levels of different proteins were quantified.