Ice-precooled CER I (200 µl) was added to GC cells and the cells were incubated on ice for 10 min after vortex oscillation for 15 sec using nuclear and cytoplasmic extraction reagents (NE-PER) (Pierce; Thermo Fisher Scientific, Inc.). Ice-precooled CER II (11 µl) was added and incubated, then swirled and shaken for 5 sec, and incubated on ice for 1 min. The supernatant (cytoplasm) was transferred to a clean ice-precooled centrifuge tube for preservation on ice. The sediment was suspended with ice-precooled NER 100 µl, incubated on ice for 10 min, and vortex oscillated 4 times for 15 sec. After centrifugation at 16,000 × g for 10 min at 4°C, the supernatant was transferred to a clean ice-precooled centrifuge tube (cell nucleus), and then western blotting was performed.

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