PMC:2783367 / 8932-9648 JSONTXT

{"project":"2_test","denotations":[{"id":"19736547-9867824-66178338","span":{"begin":98,"end":100},"obj":"9867824"},{"id":"19736547-9867824-66178338","span":{"begin":98,"end":100},"obj":"9867824"},{"id":"T9639","span":{"begin":98,"end":100},"obj":"9867824"},{"id":"T25096","span":{"begin":98,"end":100},"obj":"9867824"}],"text":"Preparation of nuclear protein extracts\nNuclear extracts were prepared as we previously described 28. In brief, after washing twice with PBS, cells were evenly re-suspended in Buffer A (10 mM HEPES-KOH pH7.9, 1.5 mM MgCl2, 10 mM KCl, 1 mM DTT, 1 mM PMSF). After incubation on ice for 10 min, cells were mixed by vortex for 30 seconds and centrifuged at 8000g for 10 seconds. Pellets were re-suspended in Buffer C (20 mM HEPES-KOH pH7.9, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, 1 mM PMSF) and incubated on ice for 15 min before vortex. The lysates were centrifuged at 8000g at 4°C for 2 min, and the resulting supernatants were taken as nuclear protein extracts, and stored at -80°C until use."}