Cells were harvested and whole cell lysates were prepared in RIPA lysis buffer as described (7 (link)) or in MIB lysis buffer (10 (link)) supplemented with 1x protease inhibitors and 1x phosphatase inhibitors (Roche) by sonication for 2 minutes. To make ER+ PDX tumor lysates, frozen PDX tumors were cryopulverized with a Covaris CP02 Pulverizer and then protein was extracted in MIB lysis buffer with sonication. Protein concentration determination and SDS-PAGE (20 μg protein per lane) were performed as described (7 (link)). Immunoblotting of nitrocellulose membranes was performed as described (7 (link)). Primary and HRP-conjugated secondary antibodies employed are listed in the Supplementary information .
Immunoprecipitation was performed as described (7 (link)), using 2 mg of lysates from hormone-deprived T47D cells with or without E2 treatment (100 nM for 45 minutes). Lysates were incubated with 2 μg anti-HA tag antibody (Santa Cruz Biotechnology Cat# sc-7392, RRID:AB_627809) or mouse IgG (Cell Signaling Technology Cat# 61656, RRID:AB_2799613) control, followed by capture of antibody-antigen complexes with protein A magnetic beads (Bio-Rad, cat# 1614013) as described (7 (link)). Immunoprecipitated proteins, as well as 20 μg of whole cell lysates (1% inputs), were analyzed by immunoblotting.
Immunoprecipitation was performed as described (7 (link)), using 2 mg of lysates from hormone-deprived T47D cells with or without E2 treatment (100 nM for 45 minutes). Lysates were incubated with 2 μg anti-HA tag antibody (Santa Cruz Biotechnology Cat# sc-7392, RRID:AB_627809) or mouse IgG (Cell Signaling Technology Cat# 61656, RRID:AB_2799613) control, followed by capture of antibody-antigen complexes with protein A magnetic beads (Bio-Rad, cat# 1614013) as described (7 (link)). Immunoprecipitated proteins, as well as 20 μg of whole cell lysates (1% inputs), were analyzed by immunoblotting.
