FIGURE 6.

Transient transfection assay. A–H, dose response to 17β-E₂, E₁, E₃, and Gen of ERE-luciferase activity in U2OS cells transiently transfected with expression plasmids for ERα or ERβ with (solid rectangle) and without (solid circle) co-transfected SRC3 was determined as described under “Experimental Procedures.” The luciferase activity obtained at the maximal dose of 17β-E₂ with ERα or ERβ in the presence of co-transfected SRC3 was set at 100%. Data were analyzed by nonlinear regression with an equation for the sigmoidal dose response (variable slope) in GraphPad Prism 4. Each data point represents the mean ± S.D. (error bars) of three experiments performed in replicates. The table below each panel shows the concentrations of the ligand (nm) at 50% of maximal response (EC₅₀) in mediating ERE-luciferase either in the presence or absence of co-transfected SRC3 plasmid. The EC₅₀ response of ERα or ERβ to 17β-E₂ in the presence of SRC3 was set equal to 100%, and the RCPs for each ligand calculated from their respective EC₅₀ values are shown. The RLA, RCA, and RRP values measured for each of the estrogens tested (obtained from Tables 1–3) are shown in each panel. I, Western blot analysis. Whole cell lysates (50 μg) prepared from SRC3-untransfected (lane 1), SRC3-transfected plus ERα-cotransfected (lane 2), or ERβ-cotransfected (lane 3) U2OS cells were run in a SDS-PAGE and transferred to nitrocellulose membrane. Membrane was probed with antibodies against SRC3 and β-actin as described under “Experimental Procedures.”