FIG. 3.

Estrogen-dependent recruitment of endogenous Fli-I to the endogenous pS2 promoter in MCF-7 cells. (A and B) ChIP assay. MCF-7 cells were incubated with or without E2 hormone for 45 min. Extracted chromatin was incubated with antibodies against the indicated proteins, and DNA purified from the immunoprecipitated (IP) fractions was analyzed by PCR with primers for the pS2 promoter or coding region. Mouse monoclonal antibody against the Fli-I LRR region (α-Fli-N) was used at 0.5 or 1 μg per assay, rabbit antiserum against a peptide from the gelsolin-like region of Fli-I (α-Fli-C) was used at 5 μl per assay, and all other antibodies were used at 1 μg per assay, including normal mouse or rabbit IgG. The results presented here are representative of seven independent experiments. (C) Simultaneous binding of pS2 promoter by ER and several coactivators. Chromatin from hormone-treated or untreated MCF-7 cells was immunoprecipitated first with anti-ER antibody; immunoprecipitates were eluted from the beads and were immunoprecipitated again with antibodies against the specified proteins. Amounts of antibodies used: 0.5 μg of α-Fli-N, 5 μl of α-Fli-C, and 1 μg of all other antibodies. DNA purified from the immunoprecipitated fractions was analyzed by PCR with primers for the pS2 promoter. (D) Simultaneous binding of pS2 promoter by Fli-I and ER or other coactivators. MCF-7 cell chromatin (E2 treated or untreated) was immunoprecipitated first by 1 μg of α-Fli-N; immunoprecipitates were eluted from the beads and were immunoprecipitated again with 1 μg of antibodies against the specified proteins. DNA purified from the immunoprecipitated fractions was analyzed by PCR with primers for the pS2 promoter.