Functional characterization of ERβ-expressing MCF-7 cells. (A) Nuclear translocation of ERα and ERβ shown by western blot analysis on cytosolic (c) and nuclear (n) protein extracts, prepared from wt MCF-7, Nt-ERβ and Ct-ERβ cells after treatment with either 17β-estradiol (10-8M, +E2) or vehicle alone (-E2) for 45 minutes. The amount of α-tubulin was also analyzed to verify the absence of cytosolic contaminants in the nuclear fractions. (B) The transcriptional activity of ERα, Nt-ERβ and Ct-ERβ was measured by transient transfection in SKBR3 cells (left) and the ability of tagged ERβ to interfere with ERα activity was assessed by comparing estrogen effects in wt, in Nt-ERβ and Ct-ERβ MCF-7 cells (right); in all cases transiently transfected ERE-tk-luc was used as reporter gene. (C) Proliferation of wt MCF-7, Nt-ERβ and Ct-ERβ cells was measured by stimulating hormone-starved cells with 10-8M E2, followed by cell counting with a colorimetric assay at the indicated times.