Cooperation between Brn-3b and ER can stimulate promoter activity. (a) Schematic diagram showing position of Brn-3 consensus binding sites and oestrogen response element (ERE) site relative to the proximal -278TATA, (now designated +1). (b) Promoter activity following cotransfection of the BSX reporter construct with Brn-3b, Brn-3a, ERα or ERβ alone or in different combinations into MCF-7 cells. Values have been equalised with internal Renilla control [renilla reporter gene driven by minimal tyrosine kinase (TK) promoter] and represent means ± SD of three independent experiments. (c) Brn-3b promoter activity when coexpressed with Brn-3b and ERα, alone or together, in cells grown in oestrogen-depleted medium and either untreated or treated with estradiol, after transfection. Grey bars show inducibility in the absence of estradiol, and black bars demonstrate the effects of adding estradiol after transfection with the constructs described. (d) Schematic showing the modified expression construct, BSXE1E, in which the Brn-3b promoter drives expression of the Brn-3b gene upstream of luciferase reporter (L). (e) Immunoblot showing changes in Brn-3b protein produced from the BSXE1E expression construct when cotransfected with Brn-3b or ERα alone or in combination (at different ratios of Brn-3b:ER, 1:1 to 1:4). Actin immunoblot was used to indicate variation in protein loading. To demonstrate that Brn-3b protein levels was dependent on the ratio of Brn-3b to ER, shorter exposure times and therefore smaller increases in Brn-3b expression are not evident when ER only is expressed. (f) Immunoblot to show increases in ER protein following transfection with expression constructs (top immunoblot) and a corresponding changes in endogenous Brn-3b protein that were dependent on concentrations of ERα (middle immunoblot). GAPDH immunoblot is included to show variation in protein loading.