Protein Extraction and Western Blot Analysis

Total proteins were extracted from fracture callus tissue or ATDC5 cells by using a Protein Extraction Kit (P0013, Beyotime, Shanghai, China) and the concentration was measured by a BCA Protein Assay Kit (P0012S, Beyotime, Shanghai, China). Western blotting was performed according to a previous study (Wang et al., 2019 (link)). Equal amounts of total protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and transferred to polyvinylidene difluoride (PVDF) membranes. After being blocked with 5% skim milk solution and then incubated with diverse primary antibodies at 4 °C overnight, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse or goat-anti-rabbit antibodies (KPL, United States). Bands were visualized using ECL Western blotting Detection kit (P0018S, Beyotime, Shanghai, China) and analyzed using ImageJ software (NIH, United States). The list of primary and secondary antibodies is shown in Supplementary Table S2.