2.5. Preparation of cytosolic and nuclear extracts from MEFs

All extraction procedures were carried out at 4°C. About 2–10 × 10⁶ cells were recovered by centrifugation at 500 × g for 5 min and washed twice with cold PBS. Cell pellets were resuspended in a hypotonic buffer (10mM HEPES pH 7.9 containing 1.5mM MgCl₂, 10mM KCl, 0.5mM DTT, 1 mM PMSF, 20 μg/ml CLAP and phosphatase inhibitor cocktail 2 and 3 at a 1:100 dilution) and incubated for 15 minutes at 4°C. NP40 was then added to a final concentration of 0.5% and the samples were gently vortexed. Samples were centrifuged at 2,800 × g in a tabletop microfuge to separate nuclei-enriched pellets from crude cytosolic supernatants. These supernatants were withdrawn and centrifuged again at 135,000 × g in a Beckman Optima TLX Ultracentrifuge (Beckman Coulter) to obtain clean cytosolic fractions. The nuclei-enriched pellets were resuspended in a high-salt buffer (20 mM HEPES pH 7.9 containing 1.5 mM MgCl₂, 420 mM NaCl, 0.2 mM EDTA, 25% v/v glycerol, 0.5 mM PMSF and 20 μg/ml CLAP and phosphatase inhibitor cocktail 2 and 3 at a 1:100 dilution). After incubating the nuclei for 30 minutes with vigorous agitation, nuclear extracts were recovered after centrifugation for 10 minutes at 10,000 × g in a tabletop microfuge.