FIG. 3.

(A) 4HT can elevate the steady-state level of SRC-1A-LUC and SRC-3-LUC in HeLa cells. HeLa cells were transfected with pERE-E1b-CAT and expression vectors for the indicated coactivators along with either pCR3.1 hERα (+) or its empty vector, pCR3.1 (−). Twenty-four hours thereafter, cells were treated with ethanol vehicle (−), E2 (E), or 4HT (T) for 24 h and harvested for luciferase activity. (B) The AF-2 of ERα is dispensable for 4HT-induced elevation of SRC-1A-LUC and SRC-3-LUC protein levels. HeLa cells were transfected in a similar manner as described above, with the addition of an AF-2-defective mutant for ERα (L539A). Cells were treated with hormone as described above with the addition of raloxifene (R). (C) The AF-1 and DBD of ERα are required for 4HT-induced elevation in coactivator expression. HeLa cells were transfected and treated as described above with the cotransfection of expression vectors for the DBD mutant (C201H/C205H) or ERα with AF-1 deleted (179C ΔAF-1) instead of the wild-type receptor. (D) Deletion of amino acid residues 1138 to 1216 of SRC-1 blocks 4HT-induced elevation of the coactivator. Expression vectors for ERα and the wild-type FLAG-tagged SRC-1 or SRC-1 deletion mutants Δ913-979 and Δ1138-1216 were transfected into HeLa cells. Twenty-four hours later, cells were treated with E2, 4HT, or their ethanol vehicle for an additional 24 h and harvested for Western analysis.