For sample preparation, when the cell culture reached 80% confluence, the cells were collected and lysed under the conditions of rotation in the mild lysis buffer for 1 hour. For the selection of magnetic beads, Dynabeads protein A (10001D, Life Technologies) and Dynabeads protein G (10003D, Life Technologies) were used for the experiment of endogenous protein, and the antibody was incubated with protein A/G in advance according to the manufacturer’s instructions. Anti-FLAG M2 Magnetic Beads (M8823, Sigma-Aldrich) were used for the experiment of exogenous protein. Except for a small fraction of input group, the remaining supernatant was incubated with the magnetic beads gently for the night at 4°C. After removing the supernatant, beads were washed five times and 10 min each. Proteins were eluted using competitive elution of 3× FLAG fusion proteins (0.4 mg/ml; F4799, Sigma-Aldrich) and were denatured in 5× loading buffer containing SDS in boiling water for 10 min. Boiled samples can be followed by silver staining and Western blot.

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