Cbl mutants are in part restoring KD Kit-mediated signaling. (A-B) Constitutive Akt activation in Kit-KD cells expressing Cbl-70Z. The 32D cells with Kit-WT or Kit-KD were engineered to express the indicated Cbl proteins, deprived from cytokines overnight, and subsequently exposed to the indicated cytokines for 10 minutes. Western blot analyses with the indicated antibodies were performed. (C) Kit-KD is not ubiquitylated. COS-7 cells were transiently transfected with the indicated constructs together with a plasmid for HA-tagged ubiquitin. After 48 hours of transfection, cells were serum starved for 12 hours and stimulated with 50 ng/mL SCF for 10 minutes or left unstimulated. Cell lysates were prepared, and equal amounts of lysates were immunoprecipitated using anti-Kit antibody. The immunoprecipitates were resolved on SDS-PAGE and analyzed with anti-HA or anti-Kit antibodies. (D) Proliferation of Kit-KD cells expressing Cbl-70Z is dependent on Akt activation. Kit-KD cells stably overexpressing Cbl-70Z were starved from IL-3 and cells were grown in the presence of 10% FCS and 10 to 15 μM Akt inhibitor or dimethyl sulfoxide (DMSO) as solvent. Cells were counted at the indicated time points by the trypan blue exclusion method, and the data are shown as fold change of the cell number compared with the start of the experiment.