FIG. 7.

Wortmannin and the Gab1 PH domain inhibit Gab1 potentiation of EGFR signaling. (A) An expression vector for EGFR was transfected into 293T cells together with vectors encoding HA-Erk2 (right) or HA-JNK 1 (left) with (second and third lanes from left) or without (leftmost lane) Gab1. At 48 h posttransfection untreated cells or cells pretreated with 100 nM Wortmannin were lysed and kinase assays were performed following immunoprecipitation with anti-HA antibodies (top blot). Immunoblots were performed with the indicated antibodies on total cell lysates (lower blots). (B) HeLa cells were transfected with vectors encoding Gab1. At 48 h posttransfection untreated cells or cells pretreated with 100 nM Wortmannin were lysed and immunoblots were performed using antiphosphotyrosine antibodies (top blot) or anti-Gab1 antibodies (bottom blot) following immunoprecipitation with anti-Gab1 antibodies. (C) HeLa cells were transfected with vectors encoding HA-JNK 1 and increasing concentrations of the Gab1 PH domain. Cells not stimulated (leftmost lane) or stimulated with EGF (100 ng/ml) for 5 min (three rightmost lanes) and then lysed 48 h posttransfection. A JNK assay was performed as described in Materials and Methods following immunoprecipitation with anti-HA antibodies (top blot). Immunoblots of total cell lysates with the indicated antibodies are shown in the lower blots. (D) Results from panel C were quantitated by densitometric scanning of the kinase assay and plotted. The vertical scale is the measurement of the density of the bands in arbitrary units.