Co-immunoprecipitation (Co-IP)

The Pierce Co-IP Kit (cat. no. 26149; Thermo Fisher Scientific, Inc.) was used to explore the interaction between proteins, according to the manufacturer's protocol. Briefly, the HeLa cells cultured in a 10-cm plate were washed twice with pre-cooled PBS, and the PBS was discarded. Subsequently, 1 ml lysis buffer was used to fully lyse the cells, the lysate was transferred into 1.5-ml microcentrifuge tubes, centrifuged for 15 min (4°C, 14,000 × g), and the supernatant protein was taken for quantification. The total volume was made up to 500 µl with lysis buffer after 500 µg protein was taken. Subsequently, 30 µl Agarose A + G was used to pre-clear lysate at 4°C for 2 h. The pre-cleared lysate was centrifuged at 4°C for 5 min at 1,000 × g and the supernatant was removed and incubated overnight with shaking at 4°C by adding the corresponding antibody. The following antibodies were used: NUAK2 (1:10; cat. no. sc-374348; Santa Cruz Biotechnology, Inc.), CYFIP2 (1:50; cat. no. sc-134308; Santa Cruz Biotechnology, Inc.) and IgG (1:50; cat. no. sc-69786; Santa Cruz Biotechnology, Inc.), which was used as a control. After 12 h of antibody conjugation, Agarose A + G (30 µl/tube) was added and shaken at 4°C for 4 h. The supernatant was discarded after centrifugation at 1,500 × g for 5 min to obtain the sediment. The precipitate was then washed three times with pre-chilled PBS to obtain the protein sample for western blotting.