FIGURE 5.

Interaction of MEMO with ERα is required for MEMO modulation of phosphorylation of ERK1/2, AKT, and ERα as well as the expression of estrogen-responsive genes. A, mapping of the MEMO interaction regions in ERα. HEK293T cells transfected with FLAG-tagged MEMO and Myc-tagged ERα, ERα AF1, ERα DNA binding domain, or ERα AF2 were immunoprecipitated (IP) with anti-Myc followed by immunoblotting (IB) with the indicated antibodies. B–D, mapping of the regions of MEMO that interact with ERα. Purified GST or GST-MEMO deletion mutant proteins were incubated with lysates of HEK293T cells expressing FLAG-tagged ERα (B–D), ERα AF1, or ERα AF2 (C). Bound proteins were subjected to Western blot with anti-FLAG. Asterisks indicate the positions of the expected purified proteins. E, immunoblot analysis of activation of ERK1/2 and AKT, ERα phosphorylation, and estrogen-responsive gene expression in MCF7 cells transfected with FLAG-tagged MEMO, MEMO(Δ82–204), or MEMO(Δ161–204) and treated with 10 nm E₂ for 24 h.