Fig. 2. (A-E) Wild-type and 679-AA-mutant cells were lysed and lysates from unstimulated (-) cells and from cells stimulated with EGF for 10 minutes (+) were immunoprecipitated with antibodies against (A-C) human EGFR or (D,E) CBL. Immunoprecipitates were transferred to nitrocellulose filters for immunoblotting after SDS-PAGE. EGFR immunoprecipitates were divided in half and resolved on two gels. One filter was incubated with a phosphorylation-specific EGFR (pTyr1045) antibody (A), and the second with a CBL antibody (B). The filter in (B) was re-probed with an EGFR antibody as a loading control (C). (D,E) The filter with CBL immunoprecipitates was incubated with a phosphotyrosine-specific antibody (pTyr) (D), and then re-probed with a CBL antibody as a loading control (E). IP, immunoprecipitation; IB, immunoblot. Molecular size in Da on the left.