Fig. 6. Role of Src-family tyrosine kinases on the Asef phosphorylation. (A) HA-Asef-expressing A431 cells were stimulated with 25 ng/ml EGF for 2 minutes. When indicated, cells were pre-treated with 4 µM PP2 for 30 minutes or with 10 µM STI-571 for 2 hours. HA-Asef was immunoprecipitated and analyzed using anti-phosphotyrosine (pTyr) or anti-HA antibodies. (B) HA-Asef expressed alone or co-expressed with v-Src, c-Src-Y537F mutant or c-Abl, was immunoprecipitated and analyzed by SDS-PAGE and immunoblotting using anti-phosphotyrosine (pTyr) or anti-HA antibodies. (C) A431 cells were transfected with HA-Asef (WT) or HA-AsefY94F (YF) in the presence or absence of the c-Src Y527F mutant. Asef was immunoprecipitated and analyzed using antibody against phospho-Tyr94 Asef (anti-pY94Asef) or anti-HA antibody. (D) HA-Asef and HA-AsefY94F expressed in 293F cells were immunoprecipitated using anti-HA antibody, phosphorylated using purified Src protein, and analyzed by immunoblotting as indicated. (E) HA-Asef-expressing A431 cells were stimulated with 50 ng/ml EGF for the indicated period and analyzed as in C.