Estrogen Receptor Binding Assay

MCF-7:WS8 and MCF-7:5C cells were grown in 20-30 15-cm dishes, and nuclear extracts (NEs) were made. The 4x estrogen response element (ERE) DNA pulldown assays were performed; by first immobilizing four copies of the Xenopus Vitellogennin ERE sequences onto Dynabeads M280 streptavidin as described previously (39 (link)). One mg of NE from MCF-7:WS8 or MCF-7:5C cells, and 0.5 μg recombinant ERα protein (Invitrogen), were added to 4xERE-beads, with either vehicle controls (ethanol or DMSO), or E₂ (100 nM), BMI-135 (1 μM), TTC-352 (1 μM), E₄ (1 μM), BPTPE (1 μM), and endoxifen (1 μM), for a 1.5 hour incubation at 4°C. After performing three washes, the final coregulator-ERα-ERE DNA complexes were eluted from the beads in 30 μl 2x SDS-sample buffer for mass spectrometry as described previously (39 (link)). The detailed methodology is presented in supplementary materials.