PlGF augments HIF-1α binding to FLAP promoter in vitro (EMSA) and in vivo (ChIP). (A) Nuclear extracts from THP-1 cells (10 μg) were incubated with a biotinylated double-stranded oligonucleotide corresponding to the region (−179 to −159 bp) of the FLAP promoter containing the proximal HRE located at −170 to −167 bp. Where indicated, 50-fold excess of unlabeled wild-type probe (lane 3) or antibody to HIF-1α (lane 4) was added. EMSA analysis was also performed with a probe containing a mutation of the HRE (−170 bp to −167 bp, lane 5). * denotes supershifted band. Data are representative of 2 independent experiments. (B) THP-1 cells were pretreated with indicated pharmacologic inhibitors before PlGF stimulation for 4 hours. The soluble chromatin was isolated and immunoprecipitated with either HIF-1α antibody (top panel) or control rabbit IgG (bottom panel). The primers used to amplify the products flanking HIF-1α binding sites in the FLAP promoter are indicated in Table 1. The middle panel represents the amplification of input DNA before immunoprecipitation. Data are representative of 2 independent experiments. Where indicated, the vertical lines show repositioned gel lanes.