Fig. 5. Tyrosine 1028 promotes the downregulation of Erbb2 in Rat-1 cells. The stability of mutant Erbb2 receptors in the presence or absence of Y1028 was examined by pulse-chase analyses using ³⁵S-methionine labeled Rat-1 stable cell lines expressing the oncogenic versions (V664E mutation) of the Erbb2^* phosphorylation mutants. (A) Erbb2^* versus Erbb2^*-Y1028F. (B) Erbb2^*-Y1144 add-back mutant versus Erbb2^*-Y1028/Y1144 double add-back mutant. Representative gels are shown for each and the average of multiple experiments is depicted graphically as a percentage of the original Erbb2 levels remaining. (C) The subcellular localization of Erbb2 was determined by immunofluorescent staining using an anti-Erbb2 antibody (Ab4, Oncogene Science) on Rat-1 cells expressing the non-oncogenic versions of Erbb2. The image was taken on a Zeiss LSM510 confocal microscope and is representative of comparable z-plane sections through the cell. Scale bar: 10 µm. (D) The ubiquitylation status of Erbb2^* mutants expressed transiently in 293T cells were examined by immunoprecipitation using an anti-Erbb2 antibody and then blotting the top half of the membrane with an anti-ubiquitin antibody (Santa Cruz). The blots were then stripped and blotted with an anti-Erbb2 antibody to check for equal levels of Erbb2. The bottom half of the membrane was incubated with anti-Cbl antibodies (Santa Cruz) to examine Erbb2-Cbl association. The phosphorylation status of c-Cbl when co-expressed with different Erbb2^* phosphorylation site mutants was examined by immunoprecipitation of c-Cbl followed by blotting with an anti-phosphotyrosine antibody (PY20, Transduction Labs). Erbb2^*, constitutively activated (oncogenic) Erbb2; Y1028F is the point mutation; NYPD is the tyrosine-deficient Erbb2 mutant; Y1028 is the Y1028 add-back mutant.