Effect of EtOH and INK128 on translation of DLBCL cells. (A) At 48 h after cells were treated with EtOH and 24 h after cells were treated with INK128 or 20 nM rapamycin (Rap.), m7GTP cap analog pull-down reactions were performed. 4E-BP1, eIF4G, and eIF4E protein abundance were analyzed by western blotting along with the total protein levels. 10 μg of protein were used for Input and whole cell lysate blots. (B,C) Cells were either untreated (Ctrl) or treated with 20 mM of EtOH for 48 h or 40 nM of INK128 for 24 h. Cell lysates were fractionated through 10–50% linear sucrose gradients (lanes 1 through 11) and the distribution of mRNA associated with ribosomal subunits 40S and 60S, monosomes 80S and polysomes of increasing molecular weight were monitored by 245 nm absorbance. (D,E) Cells were treated as described in (B,C) and incubated for 20 min with 35S-labeled amino acids. Cell lysates were resolved by SDS-PAGE and visualized with a PhosphorImager. Nascent protein synthesis was quantified and graphed as a percentage of signal intensity relative to controls. The data shown are representative of at least three independent experiments. Quantification of the signals is expressed as the percentage of the signal intensity relative to the control group in each cell line. Graphs present the mean and standard deviation from two to five independent assays. *p ≤ 0.05.