![Fig. 8. Effect of rapamycin, wortmannin, and PD098059 on eIF-4E/4E-BP1 phosphorylation and association in MO7e cells. (A and B) Factor-starved MO7e cells were treated with GM-CSF (100 U/mL) and SLF (50 ng/mL) for 15 minutes or were pulse treated with rapamycin, wortmannin, or PD098059 for 1 hour and washed twice with culture medium before 15 minutes of treatment with GM-CSF and SLF in the presence of [32P]orthophosphate. eIF-4E (A) and 4E-BP1 (B) proteins were isolated from cell lysates using anti–eIF-4E and anti–4E-BP1 antibodies, respectively, separated by SDS-PAGE, and transferred to PVDF membranes, and the intensity of [32P] radiolabeling was determined by autoradiography. Combined treatment of MO7e cells with GM-CSF and SLF increased eIF-4E phosphorylation (A) and phosphorylation of both forms of 4E-BP1 (B). Pretreatment of cells with rapamycin, wortmannin, and PD098059 blocked the stimulatory effects of GM-CSF and SLF on 4E-BP1 phosphorylation, whereas only wortmannin and PD098059 suppressed growth factor stimulation of eIF-4E phosphorylation (A). eIF-4E and 4E-BP1 content were not altered by any of the treatments indicated (data not shown). Similar results were obtained in two additional experiments. (C) Factor-starved MO7e cells were pretreated with 50 ng/mL MIP-1α, IP-10, IL-8, or PF4 or 20 ng/mL rapamycin, 10−7 mol/L wortmannin, or 50 μmol/L PD098059 for 1 hour before combined GM-CSF and SLF treatment for 15 minutes. eIF-4E proteins isolated from cell lysates by m7GTP affinity column chromatography were separated by SDS-PAGE, transferred to PVDF membranes, and probed for eIF-4E and 4E-BP1/BP2 content using anti–eIF-4E and anti–4E-BP1/BP2 antibodies, as described in the Materials and Methods. 4E-BP1 coisolated with eIF-4E in control, quiescent cells (lane 1). Treatment of MO7e cells with GM-CSF and SLF results in the dissociation of 4E-BP1 from eIF-4E, as indicated by the lack of 4E-BP1 content in lane 2 (lower panel). Pretreatment of cells with MIP-1α, IP-10, rapamycin, wortmannin, and, to a lesser extent, PD098059 suppressed the dissociation of eIF-4E from 4E-BP1 in the presence of GM-CSF and SLF (lower panel, lanes 3, 5, 7, 8, and 9). In contrast, 4E-BP1 dissociated from eIF-4E in MO7e cells pretreated with IL-8 or PF4 (lanes 4 and 6). Similar results were obtained in two separate experiments.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/89/10/10.1182_blood.v89.10.3582/3/bl_0049f8b.jpeg?Expires=1722775516&Signature=PtVm2~VQ2BGJWFitNT2b-MGHkoS14FCJYrt8NdOR3iliDO84q9YJ~~r042TEu0R7RFZCTRaOLBJZeNOk~22Ob0KJBCBrU7Py~lCjskYHanzi1AaXaP2A2-ne6zpTiEw~GIsOKZd-9viI0jd7cRBDPQlASSR6JTE6F905F1hVohMyQKrpunFF-WnlsOqlbYwMzVjmT31X6Wol69h8GR7eKqKRQrmXUMk0K3KgqiKk5Wh5KdYGdwAF1TyJh5sGjgOzgf5NJs98pkjwhLOECYAJNHFtvPPXj10k8XA-vK06B0I4V9YRQcc~RfoLFXBIwW2pdp9GWm167eup2VykQlC1-g__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Effect of rapamycin, wortmannin, and PD098059 on eIF-4E/4E-BP1 phosphorylation and association in MO7e cells. (A and B) Factor-starved MO7e cells were treated with GM-CSF (100 U/mL) and SLF (50 ng/mL) for 15 minutes or were pulse treated with rapamycin, wortmannin, or PD098059 for 1 hour and washed twice with culture medium before 15 minutes of treatment with GM-CSF and SLF in the presence of [32P]orthophosphate. eIF-4E (A) and 4E-BP1 (B) proteins were isolated from cell lysates using anti–eIF-4E and anti–4E-BP1 antibodies, respectively, separated by SDS-PAGE, and transferred to PVDF membranes, and the intensity of [32P] radiolabeling was determined by autoradiography. Combined treatment of MO7e cells with GM-CSF and SLF increased eIF-4E phosphorylation (A) and phosphorylation of both forms of 4E-BP1 (B). Pretreatment of cells with rapamycin, wortmannin, and PD098059 blocked the stimulatory effects of GM-CSF and SLF on 4E-BP1 phosphorylation, whereas only wortmannin and PD098059 suppressed growth factor stimulation of eIF-4E phosphorylation (A). eIF-4E and 4E-BP1 content were not altered by any of the treatments indicated (data not shown). Similar results were obtained in two additional experiments. (C) Factor-starved MO7e cells were pretreated with 50 ng/mL MIP-1α, IP-10, IL-8, or PF4 or 20 ng/mL rapamycin, 10−7 mol/L wortmannin, or 50 μmol/L PD098059 for 1 hour before combined GM-CSF and SLF treatment for 15 minutes. eIF-4E proteins isolated from cell lysates by m7GTP affinity column chromatography were separated by SDS-PAGE, transferred to PVDF membranes, and probed for eIF-4E and 4E-BP1/BP2 content using anti–eIF-4E and anti–4E-BP1/BP2 antibodies, as described in the Materials and Methods. 4E-BP1 coisolated with eIF-4E in control, quiescent cells (lane 1). Treatment of MO7e cells with GM-CSF and SLF results in the dissociation of 4E-BP1 from eIF-4E, as indicated by the lack of 4E-BP1 content in lane 2 (lower panel). Pretreatment of cells with MIP-1α, IP-10, rapamycin, wortmannin, and, to a lesser extent, PD098059 suppressed the dissociation of eIF-4E from 4E-BP1 in the presence of GM-CSF and SLF (lower panel, lanes 3, 5, 7, 8, and 9). In contrast, 4E-BP1 dissociated from eIF-4E in MO7e cells pretreated with IL-8 or PF4 (lanes 4 and 6). Similar results were obtained in two separate experiments.
