Fig. 4.

Assessment of the role of Src. (A) Cells pretreated with 5 μM PP2 for 30 min or transfected with Csk plasmid were stimulated with 0.5 mM H₂O₂ for 5 min. Kinase activities were determined as described in Fig. 1 legend (*p < 0.05, **p < 0.01, ***p < 0.001). (B) Cells were lysed following treatment with 0.5 mM H₂O₂ for the time period indicated. One milligram total protein was immunoprecipitated with either anti-ras (left) or anti-v-Src (right) monoclonal antibody followed by blotting with (a) anti-phosphotyrosine antibody (shown are bands that resolve at 180 kDa); (b) anti-EGF receptor antibody; and (c) anti-c-Src antibody; std, molecular weight standard; Lys, lysate of EGF-treated A431 cell as positive control. (C) Cells were lysed following treatment with 0.5 mM H₂O₂ for 0-15 min. Lysates containing 1 mg total protein were immunoprecipitated with anti-c-Src goat antibody followed by blotting with anti-phospho-EGFR monoclonal antibody. (D) Association of the SH2 domain of Src with the EGF receptor. Cells were lysed following treatment with 0.5 mM H₂O₂ for the time period indicated. Cell lysates were mixed with GST-SH2 fusion protein conjugated with agarose beads. The pull-down products were resolved by SDS-PAGE and immunoblotted with anti-EGF receptor antibody.