Verification of the Pro-Hyp response element in the Runx2 distal P1 promoter, including the Runx2-binding site. (A) A schematic representation of the relevant regions of the Runx2 distal P1 promoter. P1, P2 indicate PCR primers used to analyze ChIP/qPCR. The positions of these primers and the size of the amplified fragments are indicated at the top of the figure. (B–E) ChIP/qPCR analysis of the Runx2 distal P1 promoter in MC3T3-E1 cells. Cells were incubated with and without 1 mM Pro-Hyp for the 48 h, followed by ChIP/qPCR analysis using a control Rabbit IgG antibody (B), Foxo1 antibody (C), Foxo1 antibody (D), or Runx2 antibody (E). Experiments were repeated three times with similar results. Data are presented as means ± standard deviation (n=3), *P<0.05. Significance was calculated using the Student’s t test.