FIG. 7.

CREB and Runx2 bind to CNS1-A. (A) Gel mobility shift assays using probes for proximal and distal CREs or OSE2 present in CNS1-A. Probes are indicated to the left of each panel, and cold competitors (200-fold excess) or antibodies are indicated above each panel. Nuclear extracts were from UAMS-32P cells. A supershifted complex of the OSE2 probe and anti-Runx2 antibody is indicated by the arrowhead. wt, wild type; mut, mutant. (B) UAMS-32P cells were treated with either vehicle or 1.5 mM db-cAMP for 6 h and then subjected to ChIP analysis using antibodies to CREB, Runx2, or control IgG (αCREB, αRunx2, and αIgG, respectively). The immunoprecipitated DNA was isolated and then amplified using a primer set centered on the OSE2 and CRE sites within CNS1A (30 cycles), a primer set encompassing the 3′ end of CNS1-B (30 cycles), or a primer set centered on the TSS (32 cycles). PCR analyses were all performed within the linear range of amplification.