Figure 7.
Phosphorylation and ubiquitination of the EGFR happens regardless of overexpression of ErbB2. (A) Cells from PAE.B2 and clone 4 were incubated with or without EGF (60 ng/ml) for 2 min at 37°C. The cells were lysed and subjected to Western blotting using antibodies recognizing phosphorylated EGFR (rabbit anti-phospho EGFR [pY1045], mouse anti-phospho EGFR [pY1068], rabbit anti-phospho EGFR [pY1086], or mouse anti-phospho EGFR [pY1173]) or a rabbit anti-Erk antibody (loading control). (B) Cells from PAE.B2 and clone 4 were transfected with HA-ubiquitin. The cells were incubated with or without EGF (60 ng/ml) for 2 min at 37°C before lysis and immunoprecipitation with sheep anti-EGFR antibody. The immunoprecipitate was subjected to Western blotting using mouse anti-HA or sheep anti-EGFR antibodies. (C) Cells from clone 4 were incubated with Rh-EGF (15 ng/ml) for 15 min at 37°C. On fixation, the cells were immunocytochemically labeled with rabbit anti-ErbB2 (intracellular domain) and mouse anti-phospho EGFR (pY1068) antibodies before confocal microscopy. Asterisk (*) indicates ErbB2 overexpressing cell. Bar, 10 μm.