FIG. 7.

Phosphopeptide mapping of ErbB-2. The indicated cell lines were deprived of phosphate and serum for 12 h prior to labeling with [³²P]orthophosphate for 4 h. Cells were left untreated (A and B) or stimulated with 1 nM EGF (C) or 1 nM NDF (D and E) for 10 min at room temperature. ErbB-2 was immunoprecipitated with rabbit antiserum 21N and subjected to SDS-PAGE. In-gel tryptic digestion of ³²P-labeled ErbB-2 was performed, and the resulting peptides were extracted. Approximately 500 cpm was spotted onto TLC plates and separated by ascending chromatography in the first dimension and electrophoresis at pH 1.9 in the second dimension.