Fig. 3. c-Cbl associates with and ubiquitinates the EGFR in cells overexpressing a dominant negative form of dynamin. (A) HA-tagged c-Cbl or HA-tagged ubiquitin were transiently overexpressed in COS-7 cells in combination with wild-type dynamin, dynamin-K44A or empty vector. Serum starved cells were stimulated with 25 ng ml-1 EGF for the indicated times. Anti-EGFR immunoprecipitates (IP) were separated by SDS-PAGE and subjected to immunoblotting (IB) to detect the EGFR, or c-Cbl and ubiquitin (anti-HA). Expression levels of c-Cbl and dynamin were determined in total cell lysates. (B) The effect of the dynamin constructs on EGFR internalization was tested in COS cells transiently expressing c-Cbl in combination with wild-type dynamin (A,C) or dynamin-K44A (B,D). Serum starved cells were incubated without (A,B) or with 25 ng ml-1 EGF for 5 minutes (C,D), fixed with MeOH and stained with antibody to dynamin followed by FITC-conjugated secondary antibody (green) and with antibody to the EGFR (polyclonal antibody RK-2) followed by Texas-Red-conjugated secondary antibody (red). Analysis was performed by confocal microscopy.
