Cbl-R420Q induces ligand-independent growth in cooperation with Flt3 and potentiates Flt3-induced signaling. (A-C) Synergistic induction of ligand-independent proliferation and survival by Flt3-WT + Cbl-R420Q depends on Flt3 kinase activity. The 32D cell lines expressing the indicated constructs were starved overnight and proliferation, viability, and thymidine incorporation were analyzed as described in Figure 3. For panels A and B, results are shown as means plus or minus SD from 3 independent experiments; for panel C, the experiment shows the mean of triplicates (±SD) and is representative of 3 experiments. (D) Cbl influences Flt3-dependent signaling. The 32D-Flt3-WT cells 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. (E) Cbl proteins change the kinetics of Flt3-induced Erk activity. Cells were treated as described in panel D with the exception that they were exposed to FL for the indicated time periods. Western blot analyses using phospho-specific antibodies for Erk1/2 were performed.