Fig. 6.

Knockdown of FKBP51 increases the expression and transcriptional activity of ERα. (A and B) MCF7 (A) or MCF10A (B) cells expressing shControl or shFKBP51 were cultured in the presence of Dox for 2 d. Cells were collected and immunoblotting was performed using the indicated antibodies. The bar plot shows relative band intensities (mean ± SEM) of three independent experiments. *P < 0.05, **P < 0.01 by Dunnett t test (A) and two-tailed paired t test (B). (C) RT-qPCR analysis of ESR1 was performed in MCF7 cells expressing indicated shRNAs. Data are expressed as the mean ± SEM of three independent experiments. (D) MCF7 cells expressing indicated shRNAs were cultured and analyzed as shown in Fig. 2F. (E) MCF7 cells expressing indicated shRNAs were cultured and analyzed, as shown in Fig. 2D. The relative ratios of ERα to β-actin are reported below the image. (F) The lysates of MCF7 cells stably expressing 3× FLAG-tagged FKBP51 WT, F67D/D68V, or K352A/R356A were prepared and FLAG was immunoprecipitated. The association of ERα with FKBP51 was analyzed by immunoblotting. (G) T47D cells expressing indicated shRNAs were cultured in medium containing charcoal-stripped serum and Dox for 1 d. The cells were transiently transfected with ERE-luciferase reporter plasmids. After 24 h of transfection, the cells were treated with 10 nM E2 for another 24 h and luciferase activity was measured. Data are expressed as the mean ± SEM of three independent experiments. **P < 0.01 by two-tailed Student’s t test. CHX, cycloheximide; DMSO, dimethyl sulfoxide; IP, immunoprecipitation.