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Forty-eight hours after transfection, the cells were harvested and lysed in cold lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM PMSF, and protease inhibitor cocktail) (Beyotime, Shanghai, China) for 30 min on ice