Flt3 phosphorylates and physically interacts with c-Cbl. (A) c-Cbl is phosphorylated upon Flt3 activation. The 32D-Flt3-WT or 32D-Flt3-ITD cells stably transfected with HA-tagged Cbl-WT or Cbl-70Z were starved from IL-3 for 12 hours, and were subsequently exposed to Flt3-ligand (FL) for 10 minutes where indicated. Cbl proteins were immunoprecipitated by anti-HA antibodies and immunoprecipitates were resolved on SDS-PAGE. The phosphorylation of c-Cbl was analyzed using a phosphotyrosine-specific antibody (4G10) and the amount of total immunoprecipitated c-Cbl was analyzed by reprobing the membranes with an anti-HA antibody. (B-C) Flt3 physically interacts with c-Cbl. 32D-Flt3-WT or 32D-Flt3-ITD cells expressing endogenous c-Cbl were starved overnight (B). The next day, cells were exposed to FL as indicated. Flt3 was immunoprecipitated using anti-Flt3 antibody (as control rabbit IgG was used). Coimmunoprecipitation of Cbl was analyzed using anti-Cbl antibody. (C) 32D-Flt3-WT or 32D-Flt3-ITD cells were stably transfected with HA-tagged c-Cbl or Cbl-70Z. Cells were treated as in panel B. Cbl was immunoprecipitated using anti-HA antibody (as control mouse IgG was used). Coimmunoprecipitation of Flt3 was analyzed using anti-Flt3 antibodies. (D-E) Cbl phosphotyrosine-binding (PTB) domain interacts with Flt3-WT in vitro. (D) 293T cells were transfected with Flt3-WT and lysed after 48 hours. The lysates were incubated with the indicated GST-fusion constructs coupled to sepharose beads. The precipitated proteins were then resolved by SDS-PAGE and immunoblotted with anti-Flt3 antibodies. Equal loading of the GST-fusion proteins was confirmed by staining the membranes with Ponceau S. (E) Cell lysates prepared from 293T cells transfected with the indicated plasmid constructs were resolved by SDS-PAGE and transferred onto PVDF membranes. The membranes were then incubated with purified GST-Cbl-PTB fusion protein and the binding of GST-fusion proteins was detected with anti-GST antibodies. EGFR was used as positive control.