Protein extraction - nucleus and cytoplasm
Culture media were removed from 6-well plate, and the cells were scraped
in cold PBS with 0.5mM PMSF and centrifuged at 4°C for 2 min at 13,000g.
Pellet was resuspended in lysis buffer (10 mM HEPES pH 7.9, 10 mM KCl,
1.5 mM MgCl2, 0.5 mM PMSF, 0.1 mM EDTA, 2 μg/mL
leupeptin, 2 μg/mL antipain, 30 mM NaF, 3 mM sodium orthovanadate, 20 mM
sodium pyrophosphate and 5 mM BG-P) and incubated on ice for 15 min.
NP-40 was added, and the samples were homogenized and centrifuged for
the 30s at 13,000g at 4°C. Supernatants were used for Western blotting
assay and pellets were resuspended in extraction buffer (1.5 mM
MgCl2, 20 mM HEPES, pH 7.9, 25% glycerol, 300 mM NaCl,
0.5 mM PMSF, 0.25 mM EDTA, 2 μg/mL leupeptin, 2 μg/mL antipain, 3 mM
sodium orthovanadate, 30 mM NaF, 20 mM sodium pyrophosphate and 5mM
BG-P) and kept on ice for 20 minutes. Samples were centrifuged for 20
min at 13,000g at 4°C and supernatants were aliquoted as nuclear extract
used for EMSA assay. Protein concentration was determined using the
Bradford protein reagent (BioRad) .