Figure 3. pVHL suppressed ER-α through a direct interaction. (A) Direct interaction between ER-α and pVHL. Immunoprecipitation was performed using a pVHL antibody, and the co-precipitated proteins were analyzed using the indicated antibodies. Whole-cell extracts were obtained from 293 cells transfected with the indicated vectors. (B) GST pull-down assay. Agarose-conjugated GST-pVHL was incubated with whole-cell extracts from 293 cells transfected with the indicated vectors in RIPA buffer for 4 h. PPT indicates proteins that co-precipitated with bead-conjugated pVHL, whereas Sup indicates the supernatant. (C) pVHL suppresses transcription activity of ER-α. Two hundred and ninety-three cells were transfected with ERE-Luc and pVHL or ER-α. The increase in ERE-Luc activity was clearly reduced by VHL transfection. (D) pVHL regulates endogenous ER-α transcription activity. MCF-7 cells were transfected with ERE-Luc and VHL or si-VHL for 24 h. ER-α transcription activity was determined by a luciferase activity. (E) pVHL can suppress cyclin D1 expression. After transfection with the indicated vectors for 24 h, estrogen was added for 6 h in A549 cells. Immunoblot analysis was performed using the indicated antibodies. LE and SE indicate a long exposure and a short exposure, respectively. (F and G) pVHL can suppress Snail expression. After transfection with indicated vectors for 24 h. RT-PCR was performed to determine the expression of Snail using matched specific primers.