FIGURE 3.

CD2AP and Cbl-3 are not regulators of Ret9 ubiquitination. NIH3T3 cells were transfected with either Ret51 or Ret9, along CD2AP and Cbl-3/c (indicated as +) or with neither (indicated as −). All conditions were also transfected with HA-ubiquitin. The extent of Ret9 and Ret51 ubiquitination was ascertained as in Fig. 1E (top panel). Immunoblotting with antibodies to the shared extracellular domain of Ret was performed on the immunoprecipitates to confirm that similar amounts of Ret51 and Ret9 were expressed (middle panel). Proteasome and lysosome inhibitors were applied to the cells 6–12 h before detergent extraction to stabilize the amount of Ret in each condition, as was done in Fig. 1E. Actin Westerns of the supernatants indicated that there were equal amounts of protein in each sample (bottom panel). The expression of CD2AP and Cbl-3/c were confirmed by Myc and FLAG immunoblotting, respectively, of the supernatants. This experiment was repeated four times with similar results. IP, immunoprecipitation; W, Western blot.