Fig. 5. RUNX2 interacts with the Htra1 proximal promoter in primary mesenchymal progenitor cells. (A) Schematic diagram showing the promoter region of Htra1 (-994 bp to +200 bp) with putative RUNX2 binding sites. RCCRC is the known RUNX2 consensus binding site and TGTGGT is the reverse complement of ACCACA (RCCRC). Streptavidin-agarose pull-down assay (B) reveals the interaction of RUNX2 with biotinylated Htra1 promoter probes of length 1 kb (-994 to +72) (upper panel) and proximal 0.4 kb (-399 to +72 bp) (lower panel). (C) ChIP assay was performed in differentiating primary mesenchymal progenitor cells during osteoblast differentiation at d5. ChIP assay confirms the interaction of RUNX2 protein with the proximal Htra1 promoter during osteoblast differentiation (C). TSS, transcription start site; NC, negative control IgG; WCL, whole cell lysate.