Nuclear and Cytoplasmic Extract Preparation

Nuclear and cytoplasmic extracts were prepared by using NE-PER nuclear and cytoplasmic extraction Kit (Thermo Scientific, Rockford, IL, USA) according to the instructions specified by the manufacturer. In brief, cells were harvested by centrifuging at 500 g for 5 min, followed by washing with PBS. Cells were then lysed in ice-cold cytoplasm extraction reagent I (CER I) for 10 min and ice-cold CER II for 1 min. Cytoplasmic component was extracted by centrifuging at 16,000 g for 5 min. Proteins of the nuclear material were then extracted by adding nuclear extraction reagent to the insoluble fraction with vigorous vortex per 10 min for four times and centrifugation at 16,000 g. The extracts were stored at −80°C until use.