Western Blot

Total protein of tissues and cells lines was extracted with RIPA lysates (containing protease and phosphatase inhibitors). Protein concentration was detected using a BCA Protein Assay Kit (Beyotime, China). The equal amount of protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Then, proteins were transferred onto polyvinylidene fluoride membranes (BIO-RAD, USA). After blocked by 5% skim milk for 1 hour, the proteins were then tested with the following antibodies: anti-MAPK14 (1:1000, 9218, CST), anti-P-MAPK14 (1:1000, 4511, CST), anti-RUNX2 (1:1000, 12,556, CST), anti-Tubulin (1:1000, 2128S, CST), anti-GAPDH (1:1000, 5174, CST) at 4°C overnight. The membrane was subsequently incubated with the secondary anti-rabbit antibody at 37°C for 1 hour. Finally, the luminescence system (Bio-Rad, CA, USA) and ECL luminescence reagent were used for detection (Absin Biotechnology, Shanghai, China). ImageJ software (USA) was used for quantitative analysis. Protein abundance was normalized with tubulin or GAPDH.