Figure 2

Hormone treatment enhances AR protein binding to the VEGF promoter in chromatin of LNCaP cells. (A) Schematic diagram of the VEGF promoter showing the location of predicted ARE binding sites and primers used to amplify the specific regions of the promoter. (B) ChIP assays were performed with primers specific for the ARE I region of the VEGF promoter using chromatin prepared from LNCaP cells treated for 24 hours with either 0nM R1881 or 5nM R1881, following overnight serum starvation. Standard endpoint PCR was performed as described in text. Lane 1 shows amplification of input chromatin that was not immunoprecipitated with antibody, lane 2 chromatin immunoprecipitated with anti-pol II antibody (Upstate), lanes 3 and 4 chromatin from cells treated with 0nM or 5nM R1881 and immunoprecipitated with anti-AR (Santa Cruz) antibody and lane 6 is the negative control precipitated with IgG (Upstate). Chromatin amplified in lane 3 was obtained from cells treated with vehicle (DMSO) only. (C) PCR was performed using chromatin as described in (B) and primers specific for the ARE II region (shown in A). Lane 1 is the no DNA control, lane 2 is input diluted 1:10, lane 3 is undiluted input, lanes 4 and 5 chromatin from cells treated with 0nM or 5nM R1881 and immunoprecipitated with anti-AR antibody, and lane 6 is the IgG negative control precipitation. Chromatin amplified in lane 4 was obtained from cells treated with vehicle (DMSO) only. (D) PCR was performed using chromatin as described in (B) and primers specific for the ARE III region. Lanes are the same as in (C). (E) Quantification of immunoprecipitation was performed by SYBR Green qRT-PCR using primers described in (A). Chromatin was immunoprecipitated with anti-AR antibody from LNCaP cells treated with 0nM or 5nM R1881 as described above. Average Ct values of immunoprecipitated chromatin were normalized to input and normalized values from 5 nM R1881 treated cells are shown relative to untreated cells (0nM R1881).