The cells were harvested and incubated in NP40 lysis buffer on ice for 30 min. The lysates were centrifuged at 14,000 rpm for 10 min to remove cell debris. The supernatant was then re-centrifuged at 3000 rpm for 3 min, and mixed with 1.5 μg antibody. After 1 h of incubation, protein A/G agarose beads (Beyotime, Shanghai, China) were added. After rotation overnight at 4 °C, all beads were recovered and rinsed 3 times with NP40 buffer, followed by adding 20 μl of cracking buffer and 50 μl of loading buffer, and then incubated at 95 °C for 10 min.

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