Af0069

The protein was extracted using a Total Protein Extraction Kit (Beyotime, Shanghai, China), and the concentration was measured using a BCA Protein Assay Kit (Beyotime, Shanghai, China). The proteins were separated and transferred to PVDF membranes. The PVDF membranes were probed with the primary antibodies and the secondary antibodies. Finally, the expression of protein was measured using the ECL Western Blot kit (Biosharp, Hefei, China) and analyzed by a ChemiDocTM Touch Imaging System (Bio-Rad, CAL, USA).
The primary antibodies were anti-Sox18 (1:1000, DF8720, Affinity Biosciences, Changzhou, China), anti-β-catenin (1:1000, AF0069, Beyotime, Shanghai, China) and anti-GAPDH (1:3000, T0004, Affinity Bio-sciences, Changzhou, China). The secondary antibodies were HRP goat anti-rabbit IgG (1:1000, AS014, ABclonal, Wuhan, China) and HRP goat anti-mouse IgG (1:1000, AS014, ABclonal, Wuhan, China).

The tibias were minced and homogenized with RIPA lysis buffer and protease inhibitor cocktail (Beyotime) at 4 °C to obtain 1:9 (W/V) whole homogenate. The homogenates were centrifuged at 8000× g for 10 min under 4 °C to obtain the femoral proteins. Aliquots of protein extract were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane, operating on a Bio-rad instrument (Hercules, CA, USA). The membrane was incubated with primary antibodies against GSK-3β (AG751, Beyotime), phospho-GSK-3β (Ser 9, AF1531, Beyotime), β–Catenin (AF0069, Beyotime), Runx2 (D1L7F, CST, Danvers, MA, USA), Nrf2 (AF7623, Beyotime), HO-1 (AF1333, Beyotime), and the loading control β–actin (2148, Abcam) or α/β–tubulin (2148S, CST). Horseradish peroxidase-conjugated secondary antibody (Beyotime) was used to enhance ECL chemiluminescence detection. The protein bands were scanned and analyzed by ImageJ software (NIH, Bethesda, MD, USA). Each protein band was normalized to its β–actin band, and the levels of phosphorylated proteins were normalized to the levels of their corresponding total proteins. The results were calculated as percentage change to the ND group.