Figure 3.

BMPR2 activated the RUNX2 gene expression by binding pSmad1/5 to the RUNX2 promoter. A. The plasmid pM/BMPR2 was co-transfected after 48 h into chondrosarcoma cell lines with plasmids encoding RUNX2 promoter and renilla luciferase. B. The ChIP assay. Protein-DNA complexes from NDCS-1 cell were incubated with pSmad1/5 antibody. The binding of RUNX2 DNA was detected by PCR amplification using RUNX2-specific primers. IgG antibody and GAPDH primers were used in this assay as negative controls. C. The super gel shift assay showed the binding activity of pSmad1/5 with RUNX2 DNA in nuclear extracts of chondrosarcoma cells. For experimental controls, the labeled RUNX2 probes were incubated alone, with nuclear extracts, in combination with nuclear extracts and unlabeled probe, or in combination with nuclear extracts and IgG antibody. Data are presented as mean ± S.D. (n = 3). *P < 0.05.