Effect of chemokine and cytokine treatment on the phosphorylation of 4E-BP1 in MO7e cells. (A) Factor-starved MO7e cells were treated with SLF (lane 2), GM-CSF (lane 3), or SLF + GM-CSF (lane 4) for 1 hour or with MIP-1α (lane 5), PF4 (lane 6), IP-10 (lane 7), or IL-8 (lane 8) for 1 hour before cotreatment with GM-CSF and SLF for 1 hour. 4E-BP1 proteins were immunoprecipitated from cell lysates using anti–4E-BP1 antibodies, separated by 12% SDS-PAGE, and transferred to PVDF membranes. 4E-BP1 protein content was determined as described in the Materials and Methods. 4E-BP1 proteins were visualized upon exposure of ECL-treated membranes to film. 4E-BP1 appears as two protein bands migrating with an approximate molecular weight of 16 to 17 kD, as indicated by the position of the arrow. At left is the position of the molecular weight markers. (B) Factor-starved MO7e cells were cultured with [³²P]orthophosphate in phosphate-free RPMI. Treatments are the same as those described in (A). Control cells (lane 1) received vehicle alone. 4E-BP1 proteins were immunoprecipitated from cell lysates using anti–4E-BP1 antibodies, as described in the Materials and Methods. Proteins were separated by 12% SDS-PAGE and transfered to PVDF membranes, and the intensity of ³²P-labeling was visualized by autoradiography. At left is the position of the molecular weight markers. Protein content was determined by probing PVDF membranes with anti–4E-BP1 antibodies and visualization with ECL reagents, as described in the Materials and Methods (data not shown). (C) Scanning analysis of autoradiograms for determination of phosphorylation level for both forms of 4E-BP1. Phosphorylation levels of the larger form of 4E-BP1 were significantly higher than control levels for all treatment groups tested (P < .05). Phosphorylation levels corresponding to the smaller form of 4E-BP1 (*) were significantly higher than controls for the GM-CSF and SLF + GM-CSF treatment groups (P < .05). For both forms of 4E-BP1, phosphorylation levels evoked by combined GM-CSF and SLF treatment (**) were significantly higher than levels corresponding to the GM-CSF, SLF, MIP-1α + SLF + GM-CSF, and IP-10 + SLF + GM-CSF treatment groups (P < .05). Each bar represents the mean ± the standard deviation for three determinations obtained in separate experiments. Phosphorylation levels for each band were normalized to 4E-BP1 protein content before statistical analysis. Treatment groups are the same as described in (A). (□) 4E-BP1, larger form; (▪) 4E-BP1, smaller form.