FIG. 2.
BRG-1 associates with ER in a manner that is both hormone and AF-2 dependent. (a) MCF-7 cells were metabolically labeled, and a crude whole-cell lysate was prepared. Wild-type (wt) GST and the two ER HBD fusion proteins depicted were immobilized on glutathione-linked Sepharose (GSH-Ag) and used as affinity matrices, in the presence and absence of estrogen, to enrich for factors from the MCF-7 radiolabeled lysate. Retained fractions were boiled in an SDS-containing buffer, and the eluted fraction was diluted and subjected to immunoprecipitation (IP) with a mouse monoclonal antibody raised against hSRC-1(381-1360). The retained fractions were resolved by SDS-PAGE on a 7.5% gel and detected by radiofluorography. Sizes are indicated in kilodaltons. (b) Similar GST pulldown-reimmunoprecipitation experiments were done using a rabbit polyclonal raised against hBRG-1. (c) SRC-1 interacts directly with CBP but not with BRG-1. Flag-tagged CBP and Flag-tagged BRG-1 were expressed in a baculovirus system and purified by anti-M2 affinity chromatography. Both proteins were resolved by SDS-PAGE on a 7.5% gel, transferred to a solid support, and probed with 32P-GST-SRC-1(381-1360) in a far-Western assay. RRASV, recognition sequence for heart muscle kinase.