Cells were harvested and lysed with RIPA lysis buffer (Beyotime, P0013K) containing 1 mM protease inhibitor cocktail (Bimake, B14001) on ice for 40 min. Then, the cells were centrifuged at 12,000 rpm for 30 min at 4°C. Protein lysates were incubated with 80 μL of Protein A/G Magnetic Beads (Bimake, Shanghai, B23202) after preclearing the beads for 1 h. Then, 8 μg of anti-FLAG antibody (SIGMA, F7425) were added, and mix was subjected to gentle rotation overnight at 4°C. Western blots were operated after the beads were washed six times with lysis buffer.

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