IL-3 deprivation results in a reduction of MDM2 levels. (A) FDM cell lines of the indicated genotypes were deprived of IL-3 over a 48-hour time course. Lysates were prepared, and proteins were resolved on SDS-PAGE. Western blots were probed with antibodies to MDM2 and β-actin (loading control). (B) FDM cell lines were deprived of IL-3 for 48 hours before being restimulated with 0.25 ng/mL IL-3 for 20 minutes. Lysates were prepared, and proteins were resolved on SDS-PAGE. Western blots were probed with antibodies to pMDM2, P-AKT-Ser473, P-AKT-Thr308, P-GSK3β, and βactin (loading control). (C) Bax^−/−;Bak^−/− FDM cells were infected with a lentiviral system that allows inducible expression of constitutively active AKT1. Cells were starved of IL-3 for 48 hours (denoted as time 0) before being restimulated with 0.25 ng/mL IL-3 or treated with 4-OHT to induce expression of AKT. Lysates were prepared, and proteins were resolved on SDS-PAGE. Western blots were probed with antibodies to P-AKT-Ser473, MDM2, P-MDM2, P-GSK3α/β, and β-actin (loading control).