Nuclear and cytoplasmic protein extraction

ARPE-19 cells were collected by scraping in cold PBS and pelleted by centrifugation at 1,000 × g for 5 min at 4°C. The cell pellet was resuspended in the cell lysis buffer (including 10 mM HEPES, 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM dithiothreitol, 0.5 mM phenylmethanesulfonyl fluoride and 0.3% Nonidet P-40) and then centrifuged at 12,000 × g for 5 min at 4°C. The collected supernatant was designated as the cytoplasmic fraction. Nuclear proteins were then extracted using a buffer containing 20 mM HEPES, 25% glycerol, 1.5 mM MgCl₂, 0.6 M KCl and 0.2 mM EDTA.