Phosphorylation of CREB protein in TNBC cells. HCC1806 cells (A) and HCC70 cells (B) serum starved for 24 h (lane 1) were stimulated for 30 minutes with 10-8 M 17β-estradiol (lane 2) or pretreated for 30 minutes with 10-4 M estriol E3 (lane 3) and E3 treated cells were subsequently stimulated for 30 minutes with 10-8 M 17β-estradiol (lane 4). Cells were lysed, proteins separated in a polyacrylamide gel, blotted onto a PVDF-membrane and phospho-CREB and total CREB proteins were sequentially detected with according antibodies. Representative results of three independent preparations. (C) densitometric evaluation of Western blots results of CREB phosphorylation under various conditions normalized to total CREB expression in HCC1806 cells and HCC70.