Fig 3. Loss of E3 function in a mouse ductal carcinoma Cbl-c mutant.

(A) Cbl-c Δex7 lacks E3 activity. HEK 293T cells were transfected with plasmid encoding wild-type and mutant mouse GFP-tagged Cbl-c along with human EGFR. Triplicate plates were pooled and split at 24 h post-transfection. At 48 h post-transfection, triplicate plates were treated with 100 ng/mL EGF for 10 or 30 min prior to cell harvesting. EGFR immunoprecipitations were immunoblotted for endogenous Ub to visualize EGFR ubiquitination. The immunoblots were quantified by image analysis and the intensity of the ubiquitinated EGFR/total EGFR is shown in the graph below the immunoblot. The graph represents the average relative ubiquitination/EGFR+/- SE for 5 experiments. (B) Cbl-c Δex7 acts to prevent ubiquitination of the activated EGFR by wild type Cbl-c. HEK 293T cells were transfected with plasmid encoding wild-type and mutant mouse Cbl-c along with human EGFR and HA-tagged ubiquitin as indicated. Duplicate plates were pooled and split at 24 h post-transfection. At 48 h post-transfection, duplicate plates were treated with EGF (or water as a vehicle control) for 10 min prior to cell harvesting. EGFR immunoprecipitations were immunoblotted for HA to visualize EGFR ubiquitination. Whole cell lysates were immunoblotted and probed for mouse Cbl-c with either anti-GFP or anti-Cbl-c antibodies and human Hsc70 which served as a lysate loading control. Molecular weight standards in kDa are indicated on the left of each panel.