Figure 3.

Interactions of Mdm2 and ERα. (A) Mammalian two hybrid assay. Cells were transfected with various constructs and a GAL4-luc reporter gene and luciferase activity determined as described in the Materials and Methods. (B) Coimmunoprecipitation of Mdm2 and ERα. [³⁵S]ERα and [³⁵S]Mdm2 (Xpress-tagged) were in vitro expressed, treated with or without E2, and coimmunoprecipitated with Xpress antibodies and then analyzed by western blots as described in the Materials and Methods. (C) Coactivation of pMER by Mdm2. ZR-75 cells were treated with DMSO or 10 nM E2 transfected with pMER/GAL4-luc and Mdm2 or ECFP-Mdm2, and luciferase activity determined as outlined in the Materials and Methods. (D) Mdm2:ERα interactions - FRET anlaysis. ZR-75 cells were treated with DMSO or 10 nM E2, transfected with CFP-Mdm2 or YFP-ERα expression plasmids, and E2-dependent interactions were determined by FRET as described in the Materials and Methods. (E) FRET efficiencies. ZR-75 cells were treated with DMSO or 10 nM E2 and transfected with various constructs, and FRET efficiencies were determined as described in the Materials and Methods. Results of transfection studies (A, C and E) are means ± SE for at least 3 replicate determinations for each treatment group, and significant (p<0.05) induction is indicated (*).