Fig. 2. EGFR activation induces p120 phosphorylation at Y228. A431 cells were serum starved for 16-18 hours, then stimulated for 5 minutes with (+) or without (–) EGF (100 ng/ml). (A) Biochemical characterization of p120 mAB pY228. After EGF treatment, cells were lysed in RIPA buffer containing pervanadate. Lysates were probed by western blotting with a p120 phosphospecific mAB to Y228 (pY 228) or a general p120 mAB (pp120). EGFR immunoprecipitates were blotted with PY20 (EGFR, PY20), a general phosphotyrosine antibody, or with an EGFR monoclonal antibody (EGFR). (B) Immunofluorescent localization of Y228-phospho-p120 before (–) and after (+) EGF treatment. After EGF treatment, cells were washed for 2 minutes with PBS (containing pervanadate), then fixed with methanol. Y228-phosphorylated p120 was stained with mAb pY228 (green). Total p120 was stained with pAb F1SH (red). The individual images were merged to facilitate comparison (merge). 40x magnification. (C) Coimmunoprecipitation of Y228-phospho-p120 with E-cadherin. E-cadherin was immunoprecipitated with mAb HECD-1 before (–) and after (+) EGF treatment. Immunoprecipitates were probed by western blotting for E-cadherin (E-cad), p120 and Y228-phosphorylated p120 (pY228). Total p120 and Y228-phosphorylated p120 levels in the starting whole cell lysate (WCL) were also analyzed by western blotting.