Halt cocktail protease

Cells (1–5 million) were washed with PBS and lysed in Pierce IP Lysis Buffer (ThermoFisher, 87787) with 1x Halt Cocktail protease (ThermoFisher, 87786) for 30 minutes on ice, and centrifuged for 15 minutes at maximum speed to remove the insoluble fraction, or processed via NE-PER Nuclear and Cytoplasmic Extraction Kit (ThermoFisher, 78833) according to manufacturer’s instructions. Protein concentration was quantified via Pierce BCA Protein Assay Kit (ThermoFisher, 23225), and equal amounts of lysates were run on either SDS-PAGE 3–8% Tris-Acetate or 4–12% Bis-Tris Protein Gels (NuPAGE, ThermoFisher), then transferred to a PVDF membrane (Biorad, Wet Transfer System). Membranes were blocked for 30 minutes to 1 hour at room temperature in Odyssey Blocking Buffer/PBS (LI-COR Biosciences), and incubated with primary antibodies at 4 °C overnight. Stained membranes were washed three times in Tris-buffered saline with Tween 20 (TBS-T) and incubated for 1 hour at room temperature with secondary IRDye-conjugated antibodies (LI-COR Biosciences). Membranes were washed three times in TBS-T before imaging (Odyssey Imaging System, LI-COR Biosciences).

Reporter cells (up to 100 million) were treated with ligand (ATRA, dexamethasone) and allowed to incubate for different time points. Cells were washed with PBS with ligand added and lysed in Pierce IP Lysis Buffer (ThermoFisher, 87787) with 1x Halt Cocktail protease (ThermoFisher, 87786) in the presence of ligand for 30 minutes on ice, and centrifuged for 15 minutes at maximum speed to remove the insoluble fraction. Lysates were incubated with GFP-Trap Magnetic Agarose beads (ChromoTek, GTMA-20) overnight at 4°C on a rotator. Beads were magnetically removed and washed three times with Pierce IP Lysis Buffer with ligand added before boiling in 1x NuPAGE LDS Sample Buffer (ThermoFischer, NP0007). Blotting was done as described above. Experiments were repeated and representative images are shown.