Nuclear extracts from PBMC (n = 43) were prepared according to mini-extraction protocol (31) commonly used in our laboratory. Briefly, PBMC pellets were suspended in 500 μL of ice-cold buffer A (10 mm HEPES, pH 7.9) containing 10 mM KCl, 0.1 mM Na2EDTA, 0.1 mM Na2EGTA, 1 mM DTT and protease and phosphatase inhibitors. Cells were incubated on ice for 15 min, after which 75 μL of 10% solution of Tween 20 was added, the tubes were vortexed vigorously for 20 s and finally PBMC homogenates were centrifuged at 10000 × g for 7 min at 4 °C. After pouring off the supernatant, nuclear pellets were suspended in 150 μL of ice-cold buffer C (20 mM HEPES, 7.9 pH) containing 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT and the same protein and phosphatase inhibitors as in buffer A. Samples were then incubated at 4°C for 15 min on shaking platform followed by centrifugation at 10000 × g for 10 min at 4 °C. Obtained supernatants were used as nuclear extracts.
After determining the protein concentration by BCA Assay kit (SERVA Electrophoresis), whole cell and nuclear extracts were boiled in sample loading buffer according to Laemmli (1970) (link)) for 5 min.
After determining the protein concentration by BCA Assay kit (SERVA Electrophoresis), whole cell and nuclear extracts were boiled in sample loading buffer according to Laemmli (1970) (link)) for 5 min.