Electrophoretic mobility shift of a cAMP responsive element from cyclin D1 promoter. Cells of TNBC cell line HCC1806 (A) or of cell line HCC70 (B) were serum starved (lane 1) and either stimulated for 15 minutes with 10-8M 17β-estradiol (lane 2), or pretreated for 30 minutes with 10-4 M estriol (lane 3) before they were stimulated with 10-8M 17β-estradiol for further 15 minutes (lane 4). Cells were harvested and nuclear proteins purified from the cell pellets were incubated with the cyclin D1 specific oligonucleotide probe for 20 minutes at room temperature. After binding reaction the mixture was separated on a 6% non-denaturing polyacrylamide gel, blotted onto a nylon membrane and the biotin-labeled probe was detected using HRP-labeled streptavidin. To prove specificity of reaction a control containing a 100-fold excess of unlabeled oligonucleotide was added to a second estradiol-treated sample (lane 5) and for supershift (S) 1 μl phospho-CREB antibody was added (lane 6). Representative EMSA-assay of three independent experiments.