FIGURE 6.

Individual lobes of CaM abrogate E2-stimulated transcriptional activation by ER-α. A, HEK-293 cells were transiently transfected with both ER-α and pEGFP-CaM-F as well as pEGFP (vector), pEGFP-CaM-N, or pEGFP-CaM-C. Cells were cultured in phenol red-free medium for 24 h, then vehicle (EtOH, blue bars) or 100 nm E2 (red bars) was added to the medium. After incubation for 6 h, total RNA was isolated and quantitative RT-PCR analysis was performed to measure pS2 hnRNA. The amount of RNA in each sample was corrected for β-actin RNA in the same sample. Vehicle-treated cells were set as 1. The data represent the mean ± S.E. (error bars) of six independent experiments, each performed in triplicate. B, HEK-293 cells were transfected and stimulated with E2 as described for panel A. PR hnRNA was measured by quantitative RT-PCR. Samples were analyzed as described for pS2. The data represent the mean ± S.E. (error bars) of six independent experiments, each performed in triplicate. C, cells, transfected as outlined above, were lysed and equal amounts of protein lysate were resolved by Western blotting. Blots were probed with antibodies to ER-α (upper panel) and GFP (lower panel). A representative experiment of 4 is shown. **, p < 0.005.