Figure 7.

ERα, SMRT, and SRC-3 form a trimeric complex in vivo. A, HeLa cells were transfected with expression vector for FLAG-tagged ERα and SMRT_short and 24 h thereafter were treated with vehicle (Veh), 1 nm E2, or 100 nm 4HT for 60 min. Five percent of the cell lysate (top panel) was set aside, and remaining lysates were immunoprecipitated with Sigma's EZview Red anti-FLAG M2 affinity gel. Immune complexes were eluted with 3X FLAG peptide, and a portion of this eluant was retained (middle panel), whereas the rest was subjected to a second IP [re-immunoprecipitated (reIP)] with either SMRT antibody or rabbit IgG (bottom panel). Material from the first and second IPs as well as input samples were analyzed by SDS-PAGE and Western blotted with SRC-3 antibody. B, MCF-7 cells were treated with 10 nm E2 for 45 min and first subjected to ChIP with SRC-3 antibody, followed by re-IP with antibodies to ERα, SMRT, or IgG. Chromatin recovered from the second IP was quantitated by qPCR using primers to an amplicon in the enhancer-2 region of the cyclin D1 gene. Data represent an average ± sem of three independent experiments. Signal was not detected (n.d.) in the IgG control.