Involvement of PI3K pathway during IRES-mediated c-Myc protein synthesis. (A) Elutriated G1-arrested cells were pretreated with diluent (DMSO), 50 μM LY294002, or 50 μM PD98059 for 30 minutes at 37°C and were then incubated with 10 ng/mL (MO7e) or 1 ng/mL (F36P) GM-CSF and/or IL-3 for the indicated periods at 37°C. Total cell lysates (3 × 105 cells per lane) were separated by SDS-PAGE and were then immunoblotted. Western blotting for Akt and Erk1/2 was initially performed using antiphosphorylated form-specific antibodies followed by stripping and reblotting by antitotal Akt and Erk1/2 antibodies. These show the representative results of a minimum of 3 independent experiments. (B) Elutriated G1-arrested MO7e cells were pretreated with diluent (DMSO) or 50 μM LY294002 for 30 minutes at 37°C and were then incubated with 10 ng/mL GM-CSF for the indicated periods at 37°C. Analysis of c-myc mRNA was performed as described in the legend to Figure 1. (C) MO7e cells were labeled with radioisotope for 30 minutes at 37°C and were then incubated for the indicated periods with 10 ng/mL GM-CSF at 37°C. Analysis of the half-life of the c-Myc protein was performed as in the legend to Figure 2, except for the presence of DMSO or 50 μM LY294002 during the incubation. (D) Elutriated G1-arrested cells transfected with pSV-β-gal and pRMF were pretreated with diluent (DMSO) or 50 μM LY294002 for 30 minutes at 37°C and were then incubated with or without 10 ng/mL (MO7e) or 1 ng/mL (F36P) GM-CSF and IL-3 for 6 hours at 37°C. Both Fluc and Rluc activities were normalized to β-galactosidase activity, and luciferase activity of stimulated cells was expressed as fold activation to that of unstimulated cells. Data presented are the means (± SEM) of 3 independent experiments.
