Sc 6298

Cells were briefly rinsed with ice-cold PBS and harvested with PBS. Cells were lysed in IP150 buffer (25 mM Tris–HCl pH 7.8, 1 mM EDTA, 10% glycerol, 150 mM NaCl and 0.1% NP-40) supplemented with protease inhibitors. After preserving 10% input, total cell lysates were rotated with Flag antibody (F3165, Sigma) at 4 °C for 2 h. 30 μl of a 50% slurry of protein G–Sepharose and A-Sepharose in IP150 buffer were then added to the reaction mixtures and incubated for 1 h at 4 °C. After rapid centrifugation, the resulting Sepharose beads were washed four times with IP150 buffer, and boiled for 10 min with addition of 2X sample buffer. Co‐immunoprecipitated proteins were analyzed by SDS–PAGE followed by immunoblotting using anti‐Flag (1:5000, F3165 from Sigma), anti-HDAC1 (1:500, sc-6298 from Santa Cruz), anti‐HDAC2 (1:250, sc-7899 from Santa Cruz) or pLSD1 (1:250, ABE 1462 from Millipore) antibodies diluted in 3% BSA PBS-T for overnight. After washes with PBS-T, the membrane was incubated with the species-appropriated HRP-conjugated secondary antibody (Jackson, 115-035-003, 211-032-171) for 1 hr and then washed with PBS-T. Immuno-labelled proteins were visualized using LumiFlash Ultima Chemiluminescent substrate (Visual Protein) by LAS-4000 mini (Fuji).

The following antibodies where used in this study: RNAP II (17-672, Millipore; 61667, Active Motif; or sc-899, Santa Cruz), p-RNAP II (Ser2) (13499, Cell Signaling; 3E10, Active Motif, ab5095, Abcam), p-RNAP II (Ser5) (61085, Active Motif; 13523, Cell Signaling), H3-Ac (Upstate 07-593), H3K27me3 (07-449; Upstate), DNA-PKcs (Sc-9051, Santa Cruz), p-DNA-PKcs (S2056) (ab18192, Abcam), TRIM28 (A300-274A, Bethyl), p-TRIM28 (S824) (A300-767A, Bethyl), Tat (ab6539, Abcam), Cyclin T1 (sc-8128, Santa Cruz), p65 (sc-372 and sc-514451 Santa Cruz), CDK9 (sc-13130, sc-484), CDK7 (A300-405A, Bethyl), HDAC1 (sc-7872 and sc-6298, Santa Cruz), HDAC3 (sc-11417 and sc-376957 Santa Cruz), β-Actin (sc-47778 Santa Cruz).

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).