Cytoplasmic and Nuclear Protein Extraction

The cell pellet was resuspended in five pellet volumes of extraction buffer A (cytoplasmic extraction buffer: Combine 20 mm Tris, pH 7.6, 0.1 mm EDTA, 2 mm MgCl2, 0.5 mm NaF, 0.5 m Na3VO4. To yield ready‐to‐use extraction buffer, protease inhibitors were supplemented by adding 10 µL of 100× protease inhibitor and 10 µL of 100 mm PMSF to 980 µL of extraction buffer A shortly before use. The cells were incubated for 15 min on ice to induce hypotonic swelling of cells as a preparative step for subsequent cell lysis. Nonidet P‐40 was added to obtain a final concentration of 1% and was mixed gently by vortexing or inverting the tube. This induced cell membrane disruption and the release of cytoplasmic proteins while keeping nuclear membranes intact. Broken cells were homogenized by gently pipetting up and down three times.4 °C, 500 × g for 5 min. ≈80% of the supernatant was aspirated and transferred it to a new 1.5‐mL microcentrifuge tube (cytoplasmic extract). The residual 20% was thoroughly discarded, 10–15 pellet volumes buffer (as above) was added to the pellet of crude nuclei. The nuclei were gently resuspended by pipetting up and down and further purifying the crude nuclear preparation. The nuclei were pelleted by centrifugation at 4 °C and 500 × g for 3 min and the supernatant was roughly discarded. The rest was the nuclear fraction.