Anti p p65 antibody

Equal amounts of protein were resolved in 8% or 10% SDS-PAGE and western blotting analysis was performed as previously described [5 (link)]. Primary antibodies included anti-Hsp90 antibody (Abcam), anti-iNOS antibody (CST), anti-eNOS antibody (CST), anti-p-eNOS antibody (CST), anti-GSNOR antibody (Abcam), anti-Trx antibody (Abcam), anti-HA antibody (Santa Cruz), anti-AHA1 antibody (Abcam), anti-CDC37 antibody (Abcam), anti-IκB antibody (Abcam), anti-p-IκB antibody (Abcam), anti-p65 antibody (Abcam), anti-p-p65 antibody (Abcam) and anti-GAPDH (CST). Band intensities were analyzed by ImageJ software.

Chondrocytes were washed twice with phosphate-buffered saline (PBS) and lysed using radioimmunoprecipitation assay lysis buffer (CW Biotech, Beijing, China), and the protein concentrations were measured by a bicinchoninic acid protein assay kit (Thermo Fisher Scientific Inc., Waltham, USA). An equal amount of total protein was then separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes using β-actin as an endogenous control. The membranes were blocked with 5% skim milk for 2 h at room temperature and then incubated overnight at 4°C with primary antibodies, including anti-WISP1 antibody, anti-PI3K antibody, anti-p-PI3K antibody, anti-p65 antibody, anti-p-p65 antibody, anti-β-actin antibody (Abcam, Cambridge, UK). After washing three times with tris buffered saline with Tween-20, the membranes were incubated with the secondary antibody for 1 h at room temperature. Protein bands were visualized using the ECL system, and the optical density of the protein bands was quantified using Image J software.