500 0001edu bradford reagent

RIPA buffer (50 mM TRIS-HCl [pH 8.0], 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) with 1X protease inhibitors was used to lyse mESCs. A 100X protease inhibitor cocktail consists of 0.03 mg/mL leupeptin, 0.14 mg/mL pepstatin A, 0.02 mg/mL chymostatin, 8.5 mg/mL phenylmethanesulfonylfluoride fluoride, 33mg/mL benzamidine and solubilized in ethanol. Lysed cell suspensions were then rotated for 30 min at 4°C and were further sonicated for 10 min with a 30-s on-off cycle in a Diagenode Bioruptor Pico device to solubilize chromatin-bound proteins. Protein lysates were quantified with the Bio-Rad (500-0001EDU) Bradford reagent and loaded on SDS-PAGE gel. After transfer to nitrocellulose membranes (VWR, 95040-108), membranes were blocked with TSBT (TBS with 0.1% Tween-20) and 5% milk and then incubated with antibodies overnight (mCherry ab167453, DUX (used in (37 (link)))and signals were detected using HRP-conjugated secondary antibodies and Western Lightning Plus-ECL, Enhanced Chemiluminescence (Perkin Elmer Health Sciences, NEL 105001EA).

1 million mESCs were grown for 24 hours on 100mm dishes and then induced with doxycycline (0.25 ug/mL) for 12 hours. Cells were trypsinized, rinsed once with PBS, and lysis buffer (2 mM Tris-HCl, pH=8.0, 137 mM NaCl, 1% NP-40, 2 mM EDTA, 1x protease inhibitors) was added to lyse cells. Cells then rotated for 30 min at 4°C and were further sonicated for 10 min with a 30-s on-off cycle in a Diagenode Bioruptor Pico device to solubilize chromatin-bound proteins. Protein lysates were quantified with the Bio-Rad (500-0001EDU) Bradford reagent. 3X FLAG Peptide-coupled beads (Sigma Aldrich, F4799-4MG) were pre-washed with lysis buffer, 1 mg of protein was added to 40 μL of pre-washed beads, and samples were incubated at room temperature for 4 hours. Beads were pelleted by low-speed centrifugation. Samples were washed 5X with wash buffer (10 mM TRIS [pH 7.4], 1 mM EDTA, 1 mM EGTA; pH=8.0, 150 mM NaCl, 1% Triton X-100, 0.2 mM sodium orthovanadate, 1x protease inhibitors [see above]), boiled at 95°C in 30 μL 4X Laemelli buffer, and run on an SDS-page gel for western blotting analysis.