Fig. 2. c-Cbl associates with and ubiquitinates the EGFR at 0°C. HA-tagged ubiquitin and HA-tagged c-Cbl were transiently overexpressed in COS-7 cells. Serum starved cells were stimulated with 25 ng ml^-1 EGF for the indicated time periods on ice or at 37°C. Immunoprecipitates (IP) of the endogenous EGFR (mAb 528) and total cell lysates were separated on 6% SDS-polyacrylamide gels. Immunoblotting (IB) was used to detect EGFR (polyclonal antibody 282.7), c-Cbl and ubiquitin (both anti-HA).