Figure 7

AR and Sp1 form a complex and bind to the VEGF core promoter. (A) Quantitation of AR and Sp1 immunoprecipitated chromatin was performed by qRTPCR amplification of the VEGF core promoter region following ChIP assay of 22Rv1 cells treated with 0nM or 5nM R1881 for 48 hours prior to harvest, as described in Figure 2. Chromatin was immunoprecipitated with AR (Santa Cruz) or Sp1 (Upstate) antibodies, or IgG (Upstate), then quantified by SYBR-Green qRT-PCR using primers specific for the VEGF core promoter region (as described in text). Values shown are fold enrichment of Ab specific immunoprecipitations compared to IgG controls. (B) The interaction of AR and Sp1 in hormone treated 22Rv1 cells was demonstrated by co-immunoprecipitation of cells treated with 0nM or 5nM R1881 for 48 hours prior to harvest. Nuclear extracts were incubated with either AR Ab (Santa Cruz), Sp1 Ab (Upstate), or serum IgG and the complexes were collected by sepharose magnetic beads (Active Motif, CA). Proteins eluted from the beads were denatured and electrophoresed, then blots were probed with AR Ab. Top and bottom panels show proteins immunoprecipitated from cells treated with 0nM or 5nM R1881, respectively.