N cor sc 1609

Kasumi-1 and K562 cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and maintained in RPMI-1640 media supplemented with 20% FBS. AML1 antibodies were purchased from Cell Signaling Technology, Inc. (4334, Danvers, MA), Abcam (50541, Cambridge, MA), EMD Millipore (PC285, Billerica, MA), and Active Motif (39000, Carlsbad, CA). The Cell Signaling Technology and Active Motif AML1 antibodies gave robust AML1 pulldowns and displayed significant ChIP-PCR enrichments of control genomic regions. The Cell Signaling Technology AML1 antibody recognizes the N-terminal region of AML1, thus pulling down both wildtype AML1 and AML1-ETO. In contrast, the Active Motif AML1 antibody recognizes the C-terminal region of AML1 and does not pull down the AML1-ETO fusion protein. Therefore, the Active Motif antibody was used for AML1 ChIP-seq library preparations. An ETO antibody, PC283, was purchased from EMD Millipore (Billerica, MA). N-CoR (SC-1609) and p300 (SC-585) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). A histone H3 (tri methyl K4) antibody (ab1012) was purchased from Abcam (Cambridge, MA) and a histone H3 (tri methyl K27) antibody (ABE44) was purchased from EMD Millipore (Billerica, MA).

MCF7 and T47D were purchased from ATCC. HEK293FT was purchased from Life Technologies. Tamoxifen-resistant MCF7 clones were generated by dose-escalation 4OHT-treatment in phenol red-free RPMI1640 culture medium supplied with 10% charcoal-stripped FBS (Life Technologies): 200 nM for 2 weeks, 500 nM for 2 weeks and then 2 μM for 4 weeks. Single clones were picked up under microscope and then expanded in 2 μM 4OHT-containing phenol red-free RPMI1640 culture medium supplied with 10% charcoal-stripped FBS. Antibodies used in this report were: COPS5 (A300-014A, Bethyl Laboratories), NCoR (PA1-844A, Thermo Scientific) for western blot (WB) and IHC, NCoR (sc-1609, Santa Cruz) for IP and ChIP, ERα (SC-543, Santa Cruz) for WB, ERα (61035, Active Motif) for ChIP, GPS1 (ab4535, Abcam), CSN2 (PA1-24686, Thermo Scientific), CSN6 (sc-47965, Santa Cruz), NEDD8 (Y297) (ab81264, Abcam), PCAF (sc-13124, Santa Cruz), HDAC1 (sc-6298, Santa Cruz), HDAC3 (sc-17795, Santa Cruz), HA-tag (MMS-101P, Covance), His-tag (sc-803, Santa Cruz), actin (A-3853, Sigma), tubulin (T6074, Sigma) and secondary antibodies were purchased from Sigma. Compounds used were Tamoxifen (T5648, Sigma), 4OHT (94873, Sigma), E2 (E8875, Sigma), fulvestrant (I4409, Sigma), flavopiridol (S2679, Selleckchem) and MG132 (S2619, Selleckchem).