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Cells were lysed and incubated with 4E-BP1 antibody overnight at 4 °C. Protein G Sepharose 4 Fast Flow (Sigma-Aldrich) was added, and the solutions were gently mixed. 4E-BP1 was immunoprecipitated from 12,000×g supernatants. Samples were subjected to Western blotting using eIF4E antibody to assess the association of 4E-BP1 with eIF4E. The results were normalized to the amount of 4E-BP1.
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