Protein Expression Analysis of rBMSCs

The rBMSCs were cultured in different culture medium for 3 days. For total protein extraction, rBMSCs were lysed in ice-cold RIPA buffer and centrifugated at 12 000 rpm for 10 min at 4 °C.^{28 (link)} The amount of proteins was quantified using a BCA protein assay kit (Beyotime, China). By sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, Epizyme, China), the protein samples (10 μg) were separated and transferred to 0.22 μm pore-sized poly-vinylidene difluoride membranes (PVDF, Sangon, China). After being blocked with 0.1% tween containing 5% nonfat dry milk solution for 1 h at room temperature, the membranes were incubated overnight at 4 °C with anti-p-ERK1/2, ERK1/2 (1 : 1000, Abcam, UK), anti-RUNX-2 (1 : 1000, CST, USA), anti-GAPDH (1 : 1000, Boster, China) in the blocking buffer. After being incubated with the secondary antibodies for 1 h, specific protein bands were detected using an enhanced chemiluminescence detection system (Millipore, USA). For western blot analysis, GAPDH protein expression was used as an endogenous control for normalization.^{29 (link)}