Figure 5.

A, HESCs were transduced with recombinant adenovirus-expressing FLAG-ESR1. The next day, whole-cell protein lysates were immunoprecipitated with MED1antibody or IgG control. Immunoprecipitated protein complexes were probed with ESR1 antibody. B, HESCs were transduced with recombinant adenovirus-expressing FLAG-ESR1 for 24 hours. The next day, cells were treated with E+P or E+P+cAMP for 3 hours. Whole-cell lysates were immunoprecipitated with anti-FLAG beads. Immunoprecipitated complexes were probed using antibodies against MED1 and ESR1. The experiment was repeated three times and representative data are shown. C, HESCs were transduced with recombinant adenovirus harboring FLAG-tagged ESR1 for 24 hours. Samples were treated with E+P+cAMP for 3 hours. Whole-cell lysates were immunoprecipitated with a MED1 antibody. The immunoprecipitated complexes were probed by Western blot using antibodies against ESR1 and thyroid hormone receptor associated protein 150 (TRAP150) as indicated in the figure. The TRAP150 polypeptide, an integral component of the mediator complex, serves as a control for immunoprecipitation. The figure is the representative of two independent experiments. D, HESCs were transduced with recombinant adenovirus harboring FLAG-tagged ESR1 for 24 hours. Samples were treated with E+P+cAMP for 1 hour. ChIP was performed as described in Material and Methods. Chromatin enrichment was quantified by real-time PCR (ChIP-rt-PCR) using primers flanking potential ESR1 binding sites upstream of (−67 kb) and within (+2 kb) the WNT4 gene. The experiment was repeated twice and representative data are shown. E, ChIP was performed under similar conditions to immunoprecipitate MED1 bound chromatin as explained in Material and Methods. Primers flanking potential ESR1 bound regulatory regions indicated as WNT4 +2kb and WNT4 −67kb, were used to quantify the chromatin enrichment. The experiment was repeated twice and representative data are shown.