Effect of constitutively active 4EBP-1 on synthesis p27Kip1 and cyclin D1. (A) MCF7-4EBP-1-5A cells were cultured with or without pon A for 2 days. The cells were then pulse-labeled for 1.5 hours with 35S-amino acids in the presence of the proteasome inhibitor MG-132 to limit degradation of newly synthesized protein. Labeled cyclin D1 and p27Kip1 were immunoprecipitated, separated by SDS-PAGE, and detected by autoradiography. (B) The levels of newly synthesized p27Kip1 and cyclin D1, as detected in A, were estimated by densitometry. The level of total protein synthesis was determined by TCA precipitation of a portion of the same extracts prior to immunoprecipitation.