Figure 2.

Thrombin does not activate the EGF receptor in Swiss 3T3 cells. Swiss 3T3 cells were left inactive (0) or were stimulated for 5 min with EGF (E5, 10 nM) or thrombin (1 U/ml) for 1 min (T1) or 5 min (T5) in the presence or absence of AG1478 (0.5 μM). Cells were lysed and extracted proteins were immunoprecipitated with (A) EGF receptor antibodies or (B) phosphotyrosine antibodies. Proteins were separated by SDS-PAGE and Western blotted for phosphotyrosine (A) and Shc (B). It can be seen that the EGF receptor was tyrosine phosphorylated when cells were acutely activated with EGF (5 min). Thrombin had no effect on the phosphorylation state of the EGF receptor (A). In all lanes there were equal amounts of EGF receptor immunoprecipitated (not shown). Similarly, EGF brought about the association of 52- and 66-kD forms of Shc with phosphotyrosine immunoprecipitates, but thrombin had little such effect (B). The heavy chain (HC) of the immunoprecipitating antibodies is indicated, as are positions of molecular weight standards (shown in kD) in B. Qualitatively identical results were seen in four separate preparations.