Runt related transcription factor 2 runx2

The cells were harvested and proteins were extracted with RIPA lysis buffer (Beyotime, Shanghai, China). The protein concentration was determined using the BCA protein Assay (Beyotime, Shanghai, China). Equal aliquots of 40 μg per sample were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10–12%) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). Following blocking in 5% nonfat dry milk (dissolved in TBST, TBS plus 0.1% Tween-20) for 1 h at room temperature, the proteins of interest were probed with primary antibodies overnight at 4°C: osteocalcin (1:1,000; cat. no. ab13418; Abcam, Cambridge, UK), collagen I (1:1,000; cat. no. ab6308; Abcam), runt-related transcription factor 2 (RUNX2;1:1,000; cat. no. 12556; Cell Signaling Technology, Inc., Danvers, MA, USA) and β-actin (1:5,000; cat. no. 3700; Cell Signaling Technology, Inc.). Subsequently, membranes were incubated with IRDye 800CW-labeled goat anti-rabbit IgG (H+L; 1:10,000; cat. no. 926-32211; LI-COR Biosciences, Lincoln, NE, USA) and goat anti-mouse IgG (1:10,000; H+L; cat. no. 926-32210; LI-COR Biosciences) for 1 h at room temperature. The blots were then visualized using an Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA). The band density was quantified using Odyssey software version 3.0 (LI-COR Biosciences) and normalized to β-actin.