2.5. Western blot analysis

BMSCs were seeded into 6-cm-diameter culture dishes and cultured in complete medium. EMD with a concentration of 5, 25, 50 μg/ml was added to the experimental group, respectively. And, the same amount of complete medium was added to the control group. The cells were collected after 3 days of culture. Total proteins were extracted from the BMSCs by ice-cold RIPA lysis buffer. Subsequently, lysate proteins were resolved by electrophoresis, followed by transfer to polyvinylidene difluoride (PVDF) membranes (Millipore, USA). Then, the membranes were blocked in 5% skim milk for 1 h and incubated overnight at 4℃ in diluted primary antibody. Finally, images of the target strip were developed in the way of using a Western Chemiluminescent HRP Substrate Kit (Millipore, USA). Primary antibodies directed against Runx2 (1:1500, Rabbit mAb#12,556, monoclonal antibody, Cell Signaling Technology, MA, USA), Osterix (1:1000, ab209484, monoclonal antibody, Abcam, UK), ALP (1:1500, ab5462, monoclonal antibody, Abcam, UK), β-catenin (1:6000, ab2572, Abcam, UK) and GAPDH (1:1000, bs-2188 R, Bioss, China). ImageJ was used to analyze the gray value of each band.