Ethanol alters 4E-BP1 and eIF4E association and decreases cap-dependent translation in lymphocytes. (A) Immunoprecipitation (IP) of protein lysates with either anti-mTOR antibody or control IgG was assayed to analyze changes in association of mTOR and Raptor. (B) Raji, SUDHL-4, B cells, PBLs, MCF-7, and BT-474 cells were treated with ethanol (0-20 mM) for 10 days, and lysates were collected. Levels of p4E-BP1 and total 4E-BP1 were analyzed by Western blot. (C) A total of 250 μg total lysates was incubated with a 5′ 7mG cap analog conjugated to agarose beads and used to pull down cap-bound eIF4E. (D) Total eIF4E levels were measured by Western blot analysis. (E) Immunoprecipitation (IP) of protein lysates with either anti–4E-BP1 antibody or control IgG was assayed to analyze changes in association of 4E-BP1 and eIF4E. (F) Cap-dependent translation of ethanol-treated cells was measured by transfection with a bicistronic luciferase construct (cap-dependent firefly luciferase and IRES Renilla luciferase). Six hours after transfection, cells were treated with appropriate amounts of ethanol for a further 24 hours. Cells were then harvested, and luciferase activity was measured. Data points represent the mean of 3 independent experiments, with error bars corresponding to SD (P ≤ .002).