Effect of chemokine and cytokine treatment on eIF-4E phosphorylation in MO7e cells. Factor-starved MO7e cells maintained in phosphate-free RPMI supplemented with [³²P] orthophosphate were treated with GM-CSF, SLF, or a combination of GM-CSF and SLF or with MIP-1α (B and D), IL-8 (B), IP-10 (C and D), or PF4 (D) for 1 hour before treatment with GM-CSF + SLF for various times over a 12-hour period. Control cells (lane 1, A through E) received vehicle only. eIF-4E proteins were isolated from cell lysates by immunoprecipitation with anti–eIF-4E antibodies (A, C, and D) or by m⁷GTP column chromatography (B), 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 indicated the position of the molecular weight markers. eIF-4E appears as a single band at approximately 27 to 29 kD, as indicated by the position of the arrow. (E) eIF-4E protein content was determined by immunoblotting PVDF membranes with anti–eIF-4E antibodies and horseradish peroxidase-linked protein G. eIF-4E proteins were visualized upon exposure of ECL-treated membranes to film. Cells were pretreated with chemokines for 1 hour before 2 hours of treatment with GM-CSF and SLF.