CHO cells transfected with the indicated plasmid were lysed in radioimmunoprecipitation assay buffer containing protease inhibitors (Sigma-Aldrich, P8340) for 30 min at 4°C. Lysates were centrifuged at 10,000 rpm for 10 min at 4°C, and supernatants were transferred to new tubes and supplemented with LDS loading buffer and 5% (v/v) β-mercaptoethanol. Samples were boiled for 5 min, separated by SDS–polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride membranes. Membranes were blocked for 1 hour in 5% milk in PBST and incubated with mouse anti-Flag M2 antibody (Sigma-Aldrich) at 1:5000 dilution overnight at 4°C on a shaker. Membranes were washed three times with PBST for 5 min and incubated with Goat Anti-Mouse IgG (H+L)-HRP Conjugate (Bio-Rad, 1706516) diluted 1:5000 in 5% milk PBST for 1 hour. Membranes were washed three times in PBST for 5 min, incubated with SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific), and scanned using a C-DiGiT Blot Scanner (LI-COR).

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