Validation of the C4–12/Flag.ERβ cell line.

<p>A – Flag.ERβ stably expressed in C4–12/Flag.ERβ cells was detected (because of low expression level, the ERβ band in the input lane was faint) and immunoprecipitated with anti-Flag antibody. Lysates of MCF-7/C4–12 cells transiently expressing Flag.ERβ were used as positive controls. B – Luciferase reporter assays confirmed the expression of a functional ERβ in C4–12/Flag. ERβ cells. C4–12/Flag.ERβ cells were cultured in 48-well plates, then transfected with 3xERE-Luc and RTK plasmids. After 24 hours of treatment with 10 nM E2 or Vehicle (Ethanol), cells were lysed with Passive Lysis Buffer (Promega) and processed with Dual Luciferase Assay (Promega). (RLU = relative light units; error bars = standard deviation).