Association of Rap1A with osteoblast differentiation.

<p><b>(a)</b> Immunoblot analysis of Rap1A expression during osteogenic differentiation. The protein extracts were immunoblotted with anti-Rap1A antibody. β-Actin was used as the internal control. <b>(b-f)</b> Changes of osteoblastic markers gene expression in differentiated C2C12 cells. C2C12 cells were cultured with osteogenic medium containing ascorbic acid (50μg/ml), and β-glycerophosphate (10 mM) for the indicated times. Quantitative real-time PCR was performed for the mRNA expression of Rap1A, Runx2, Col1a1, Osteocalcin, and Osterix. <b>(g)</b> Alkaline phosphatase (ALP) staining and ALP activity measurement were determined during osteoblastic differentiation from C2C12 cells. <b>(h)</b> Osteoblastic mineralization of C2C12 cells was determined by Alizarin red staining and calcium content quantified in the cellular matrix by spectrophotometer (OD562nm). Data represent means ± SD of triplicate samples. *P<0.05, **P<0.01 vs. the undifferentiated cells.