Dynabead protein a and g

Cells were collected using lysis buffer supplemented with 1× complete protease inhibitor mix (Roche). The protein concentrations of the lysates were quantified using a BCA Protein Assay Kit (Pierce). Cell lysates were incubated with primary antibody and Dynabead Protein A and G (Life Technologies) overnight on a rotator. Immunoblotting analyses were performed as previously described (28 (link)). Proteins were separated on SDS-PAGE gels and transferred to nitrocellulose membranes. Antibodies used for immunoprecipitation and immunoblotting were p53 antibody (DO-1, sc-29435); ERα antibody (F10, Santa Cruz); ERβ antibodies (CWK-F12 produced in our lab (25 (link),29 (link)); H3K4me3 (ab8895, Abcam); H3K9me3 (ab8898, Abcam); N-CoR (sc-1609, Santa Cruz); and SMRT (sc-1610, Santa Cruz).

ChIP assays were performed as previously described (10 (link)). Experiments were conducted in hormone-depleted MCF-7 cells treated with 0.1% Control EtOH Vehicle or 10nM E2 for 45min. Chromatin was cross-linked with 1% formaldehyde for 10 min at room temperature. Cells were washed with PBS and collected using lysis buffer supplemented with 1× complete protease inhibitor mix (Roche). Chromatin was sonicated in lysis buffer to 300–500 bp size fragments. Antibodies were incubated with cell lysates overnight for chromatin collection and then with Dynabead Protein A and G (Life Technologies) for 6 h. ChIP DNA was isolated using PCR puri cation kit (QIAGEN) per the manufacturer’s instructions and used for quantitative real-time PCR. Amounts of ChIP DNA were normalized to inputs. Antibodies used for ChIP experiments were H3K4me3 (ab8895, Abcam); H3K9me3 (ab8898, Abcam); p53 (DO-1, sc-29435); RNA Polymerase II (29634A, CTD4H8 clone, Upstate); ERα (HC-20, Santa Cruz); ERβ antibodies were a combination of CWK-F12 (produced in our lab) (25 (link)), GTX70182 (GeneTex), GR40 (Calbiochem), and PA1-311 (Af nity Bioreagents); N-CoR (sc-1609, Santa Cruz); and SMRT (sc-1610, Santa Cruz).