Extraction of cytoplasmic and nuclear proteins

The cytoplasmic and nuclear proteins were prepared as previously described.26 Briefly, cells were collected for Western blot as described in Western blot analysis section. Cells were washed three times with ice-cold PBS and then suspended in hypotonic buffer for cytosolic extraction. After incubation on ice for 15 min, 12 μL of 10% (v/v) NP-40 was added to the suspension for another 10 min. The cytoplasmic fraction was prepared by centrifugation for 2 min. The remaining cell pellet generated after initial centrifugation was washed three times with the hypotonic buffer and suspended in high-salt buffer. The suspended cell pellet was incubated for 30 min on ice and then centrifuged for 10 min to obtain the nuclear fraction. Soluble cell lysates were immunoblotted with the designated antibodies.