Expression of 4EBP-1-5A inhibits proliferation of MCF-7 cells. (A) MCF7-4EBP-1-5A cells were treated with or without pon A for up to 5 days as indicated. Cell extracts representing 2 × 105 cells were used for Western blotting with anti-4EBP-1. The lower band (arrow) represents endogenous, hypophosphorylated 4EBP-1 and 4EBP-1-5A induced by pon A. (B) MCF7-4EBP-1-5A cells were cultured in the presence or absence of pon A for 2 days. Cell extracts were prepared and equal amounts of protein were incubated with 7-methyl-GTP-Sepharose as described in Experimental Procedures. Bound eIF4E and associated 4EBP-1 were detected by Western blotting. (C) MCF7-4EBP-1-5A (left) or MCF-7 (right) cells were grown in the presence (open circles) or absence (closed circles) of pon A. Cell numbers were counted on days 1, 3 and 5 as indicated. Each point represents the mean (± standard error) of either 4 (left panel) or 3 (right panel) independent determinations of cell number.