Mutation of Brn-3 site reduces the inducibility of promoter by ERα and also abolishes cooperativity. (a) Testing effects of ERα with or without Brn-3b on activity of deletion promoter, BS-SS, in which putative binding site for Brn-3b and ERα are lost. Reporter gene activity was adjusted using internal control, Renilla luciferase. Values are shown relative to BS promoter activity in the presence of control empty vector, LTR, set at 100%. Effects of ERα or Brn-3b, alone or together on the intact BS promoter or deletion (BS-SS) promoter are compared (b) Reporter gene activity following cotransfection of Brn-3b or ER (alone or together) with WT or mutant Brn-3b reporter construct in which Brn-3b site is mutated. Values have been adjusted for internal Renilla luciferase control and are expressed as percentages of activity in empty vector transfected cells (set at 100%) for the respective promoters. Results represent means ± SD from three independent experiments. (c) Similar reporter assays were used to show changes in activity of Brn-3b promoter containing mutation of ERE alone (ERE mutant) or double-mutant lacking both sites (Brn-3/ERE mutant) when coexpressed with Brn-3b +/- ERα. The data represent means ± SD of three independent experiments. (d) Representative PCR product obtained using ChIP DNA (immunoprecipitated with Brn-3b antibody) and primers to amplify and promoter region containing the putative Brn-3b binding site. Input column represents PCR amplification using one-tenth of isolated DNA before ChIP. Middle column (-ve Ab) shows product following ChIP assay with negative control α-rabbit Ab only). Right column (Brn-3b Ab) shows product resulting from ChIP assay using Brn-3b Ab.