Disruption of signal transduction of GPR30 in TNBC cell lines. Western blots (A) HCC1806 and (B) HCC70. Serum starved cells (lane 1) were stimulated for five 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 with 10-8 M 17β-estradiol (lane 4). Cells were lysed, proteins separated in a polyacrylamide gel, blotted onto a PVDF-membrane, and the indicated proteins were sequentially detected with antibodies against phospho-src (panel 1), total-src (panel 2), phospho-EGF-receptor (panel 3), total EGF-receptor (panel 4), and actin, as housekeeping gene (panel 5). Representative results of three independent preparations. (C) and (D) densitometric evaluation of Western blot results of p-src and pEGFR in HCC1806 cells (C) and HCC70 (D).