MOESM5 of Identification of a pituitary ERα-activated enhancer triggering the expression of Nr5a1, the earliest gonadotrope lineage-specific transcription factor

Additional file 5. A Knockdown efficiency of ERα SiRNA in αT3–1 cells. αT3–1 cells were transiently transfected in duplicates with scramble or Erα SiRNA. Proteins were extracted 48 h later. Western blots for ERα and GAPDH immunodetection were performed as indicated in “Materials and Methods.” Top: ERα immunodetection: The 66-kDa and the 36-kDa isoforms are expressed in αT3–1 cells. Erα SiRNA allows efficient knockdown of both isoforms. Bottom: GAPDH immunodetection for normalization. B ERα specific antagonist MPP dihydrochloride modulates α enhancer cis-regulatory activity. αT3–1 cells were transiently transfected with control (Prl) or full-length α enhancer (α enh) Pluc constructs. Transfected cells were treated with either vehicle or MPP dihydrochloride at the indicated concentrations. Relative luciferase activity was measured as indicated in “Materials and Methods.” Results were normalized for control Pluc–Prl–luc and are the mean ± SEM of six independent experiments in quadruplicates. ANOVA followed by Dunnett’s multiple comparison tests was performed to compare drugs at different concentrations against vehicle condition. Significant difference with the vehicle: “c” p < 0.001.