Fig. 6.

The ER-α (S294A) mutant promotes the transcription of ER-α target genes. (A) Lentivirus-infected MCF7 cells were exposed to Dox in a medium containing charcoal-stripped serum to induce the expression of an ER-α shRNA as well as transiently transfected with a vector for shRNA-resistant FLAG–ER-α (WT or S294A) for 2 d and were then treated (or not) with 10 nM E2 for 4 h. Total RNA isolated from the cells was subjected to the RT-qPCR analysis of the indicated ER-α target gene mRNAs. The RT-qPCR data are means ± SEM from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 (unpaired t test). (B) Lentivirus-infected MCF7 cells were exposed to Dox to induce the expression of an ER-α shRNA as well as transiently transfected with a vector for shRNA-resistant FLAG–ER-α (WT or S294A) for 2 d, after which expression of ER-α target genes was determined as in A. Data are means ± SEM from three independent experiments. **P < 0.01, ***P < 0.001, and ****P < 0.0001 (unpaired t test). (C) MCF7 cells were transiently transfected with expression vectors for FLAG-tagged WT or S294A mutant forms of ER-α in a medium containing charcoal-stripped serum for 2 d and were then stimulated (or not) with E2 for 30 min, after which cell lysates were subjected to immunoblot analysis with the indicated antibodies. The relative ER-α–pS118/FLAG band intensity ratio was determined. Data are from a representative experiment. (D) MCF7 cells were transiently transfected with expression vectors for FLAG-tagged WT, S118A, or S118D forms of ER-α (or with the corresponding empty vector, Vec) in a medium containing charcoal-stripped serum for 2 d and were then stimulated (or not) with E2 for 4 h, after which cell lysates were subjected to immunoblot analysis with the indicated antibodies.