Ap0140

Conditional cultured chondrocytes and disc cells were washed with PBS and lysed with RIPA buffer containing a proteinase and phosphatase inhibitor. The cell lysates were sonicated, followed by heating and adding a loading buffer. Processed protein samples were loaded onto 10% SDS‐PAGE gels, separated by electrophoresis, and then electrotransferred to PVDF membranes. The blotting membranes were blocked with 5% nonfat milk for 1 h at room temperature and incubated overnight at 4°C with primary antibodies, including HMGB1 (1:1000; EPR3507; Abcam), RAGE (1:1000; EPR21171; Abcam), TLR4 (1:1000; 19811‐1‐AP; Proteintech), MMP‐3 (1:10000; 66338‐1‐Ig; Proteintech), NF‐κB p65 (1:1000; 8242; CST), phospho‐NF‐κB p65 (1:1000; 3033; CST), AKT (1:1000; 4691; CST), phospho‐AKT (1:1000; AP0140; ABclonal) and GAPDH (1:5000; GB11002; Servicebio). The proteins were detected with horseradish peroxidase‐conjugated secondary antibodies and visualized using Western Bright ECL (K‐12045‐D10; Advansta). The blotted protein bands were quantitatively analysed using ImageJ software.

Monoclonal anti-Actin Beta (ACTB) antibody (AC026, ABclonal Technology, Wuhan, China), monoclonal anti-GFP antibody (AE012, ABclonal Technology), monoclonal anti-RFP antibody (AE020, ABclonal Technology), monoclonal anti-His antibody (AE003, ABclonal Technology), polyclonal anti-phospho-AKT antibodies (AP0140, ABclonal Technology), polyclonal anti-phospho-FOXO antibodies (AP0684, ABclonal Technology), mouse control IgG (AC011, ABclonal Technology), rabbit control IgG (AC005, ABclonal Technology), Horseradish Peroxidase (HRP)-conjugated goat anti-rabbit IgG light chain (AS061, ABclonal Technology), polyclonal anti-AKT antibodies (WL0003b, Wanleibio, Shenyang, China), polyclonal anti-FOXO antibodies (WL02891, Wanleibio), Alkaline phosphatase (AP)-conjugated horse anti-mouse IgG (ZB2310, ZSGB-BIO, Beijing, China), and AP-conjugated goat anti-rabbit IgG (ZB2308, ZSGB-BIO).