c-Cbl facilitates ubiquitylation of Flt3. (A) Flt3 receptor is rapidly degraded in 32D cells. The 32D cells expressing Flt3-WT or Flt3-ITD receptors were starved overnight, and pretreated for 2 hours with cycloheximide before incubating with FL for the indicated time points. Cells were harvested at the indicated time points, cell lysates were resolved on SDS-PAGE, and Flt3 protein levels were determined using anti-Flt3 antibodies. The densitometric analyses were performed as described in “Materials and methods.” The closed circles (upper) depict degradation of the presumably mature form of Flt3 (160 kDa), while the open circles (lower) show degradation of the immature form of Flt3 (130 kDa). (B) Ubiquitylation of Flt3 is inhibited by Cbl-70Z. COS-7 cells were transiently transfected with plasmid constructs encoding Flt3-WT or -ITD with or without Cbl-70Z, together with a plasmid encoding HA-tagged ubiquitin. At 48 hours after transfection, cells were left unstimulated or were exposed to FL as indicated. Flt3 immunoprecipitates were resolved on SDS-PAGE and ubiquitylation of Flt3 was analyzed by immunoblotting the membranes with an anti-HA antibody.