Figure 4: Methylation-dependent interaction between ERα and PHF20 in cells.

(a,b) PHF20 interacts with ERα in cells. Western blot analysis of reciprocal co-IP experiments in HEK-293T cells co-transfected with Flag-PHF20 and Myc-ERα (a) or Flag-ERα and Myc-PHF20 (b). WCE and immunoprecipitated Flag-tagged proteins were probed with the indicated antibodies. (c) ERαK235R mutation reduces the interaction between ERα and PHF20 in cells. Western blot analysis of co-IP experiments in HEK-293T cells co-transfected with Myc-PHF20 and either Flag-ERα or the Flag-ERαK235R mutant. (d) G9a enhances the interaction between ERα and PHF20 in cells. Western blot analysis of co-IP experiments using HEK-293T cells co-transfected with Flag-PHF20 and Myc-ERα ±G9a. (e) The G9a inhibitor BIX-01294 (BIX) diminishes the interaction between ERα and PHF20 in cells. Western blot analysis of co-IP experiments using HEK-293T cells co-transfected with Flag-ERα and Myc-PHF20. Cells were treated with the indicated amounts of BIX for 24 h before being collected for co-IP experiments. Western blotting analysis of the WCE shows that the total levels of G9a, PHF20 and ERα were not affected by BIX treatment.