Figure 7

MOZ-TIF2 participates in the ER activation complex as does TIF2 and interacts with CBP in vivo. A, B, and C. For identification of the interaction between MOZ-TIF2 and CBP, HEK 293 cells were transiently transfected with His-tagged MOZ, MOZ-TIF2, and vector alone. After 36 hours, the cells were lysed and co-immunoprecipitation conducted by an anti His-tag antibody conjugated to agarose beads. The co-immunoprecipitated proteins were separated by SDS-PAGE and western analyses were performed with an anti-CBP antibody. The input represents 5% of the protein used in the co-immunoprecipitation and was from cells transfected with MOZ-TIF2. A. Western blot showing MOZ and MOZ-TIF2 bound to the agarose beads. B. Transfected cells subjected to SDS-PAGE to demonstrate the presence of CBP in the transfected HEK293 cells. C. Western blot analysis to demonstrate CBP co-immunoprecipitating with MOZ and MOZ-TIF2. D and E. To determine the presence of MOZ-TIF2 in ER activation complexes, HEK 293 cells were transiently co-transfected with pEGFP-MOZ-TIF2 or pEGFP-TIF2 and with an expression plasmid for the estrogen receptor, RSV-hER. After the addition of 50 nM estradiol for 36 hours, the cells were lysed and co-immunoprecipitation was performed with 500 μg protein using an anti-EGFP antibody. The co-immunoprecipitated proteins were subjected to 12% SDS-PAGE fractionation and ER was detected by western blot with anti-ER antibody. The input represents 5% of the protein used in the co-immunoprecipitation. D. The expression level of EGFP fusions in cells transfected with MOZ-TIF2 (GFP-MT2) and TIF2 (GFP-TIF2). E. The co-immunoprecipitation of ER by MOZ-TIF2 or TIF2. Input 1 is from cells transfected with pEGFP-TIF2 and input 2 is from cells transfected with pEGFP-MOZ-TIF2. F. Co-localization between endogenous MOZ and CBP in HEK293 cells during the cell cycle. Top panel, co-localization of MOZ and CBP in the nucleus during G1-S-G2 stage. Middle panel, the co-localization disassociated from chromosomes during the metaphase. Bottom panel, the restoration of co-localization with chromatin.