FIG. 8.

Requirement of endogenous GAC63 for ER-mediated gene expression. (A) MCF-7 cells were transfected with 200 ng of MMTV(ERE)-LUC reporter plasmid and 90 (+) or 120 (++) pmol of siRNA duplex against GAC63 or scrambled-sequence siRNA duplex. After 48 h, cells were treated with 100 nM E2 and then harvested after an additional 24 h for luciferase assays (upper panel). Cell extracts were also tested by immunoblotting using anti-GAC63 antibody; the membrane was stripped and reprobed with antibody against β-actin (lower panels). In a similar experiment, in which GAC63 expression was dramatically reduced, ERα protein levels were measured (lower panels). The results shown are representative of four independent experiments. RLU, relative light units. (B) MCF-7 cells were transfected with 120 pmol of siRNA duplex against GAC63 or scrambled-sequence siRNA duplex. After 72 h, cells were treated with 100 nM E2 or left untreated for an additional 24 h before harvest. mRNAs were analyzed by RT-QPCR to measure the levels of GAC63, pS2, and GAPDH transcripts. Results shown for GAC63 and pS2 transcripts are normalized by the levels of GAPDH transcripts and are representative of four independent experiments.