Ms 298 p

Immunoblotting/Western blotting was performed as previously described [9 (link), 23 (link), 27 ]. Membranes were probed with primary antibodies recognizing total PR (Santa Cruz Biotechnology, sc-7208 or ThermoScientific, MS-298-P), OGT (a kind gift from Dr. Gerald Hart at the Johns Hopkins School of Medicine), and O-GlcNAc (Abcam, ab2739). All Western blotting experiments were performed in triplicate, and representative experiments are shown.

For ChIP experiments, T47D cells were grown to 70–80% confluency in 15 cm² dishes in phenol red-free media containing charcoal stripped serum. The next day, cells were pretreated for 30 min with 100 n_M RA or vehicle then treated with 100 n_M P4 or vehicle for 1 h. Immunoprecipitation for PR was performed using antibody PR Ab-8 (recognizes both PRA and PRB, MS-298-P, Thermo Fisher, Grand Island, NY, USA), RARα, p300, (ab41934, ab54984, Abcam, Cambridge, MA, USA) or CBP (7389, Cell Signaling, Danvers, MA, USA). Cells were processed using the ChIP-IT Express kit (Active Motif, Carlsbad, CA, USA). Chromatin was sheared using an S220 Focused Ultrasonicator (Covaris, Woburn, MA, USA).