FIG. 7.

Downregulation with siRNA of Grb2-mediated Gab1 recruitment creates a dependence on PI3K for both ERK1-2 activation and Gab1/Shp2 recruitment in HEK293 cells maximally stimulated. (A) Cells were treated or not treated (−) with siRNA against Grb2 (Grb2-300328) or against a scrambled sequence (control). Following cell treatment with wortmannin (+W) or LY294002 (+LY) or no treatment (−) and then stimulation with EGF (50 ng/ml) as shown, lysates were subjected to immunoblotting with the indicated antibodies. (B) Cells were transfected with Grb-2300328 or control siRNA and then treated with PI3K inhibitors and EGF (50 ng/ml), as described above. Cell lysates were then processed for Gab1 immunoprecipitation (IP Gab1), followed by anti-pTyr and anti-Gab1 immunoblotting, as indicated. For the middle and bottom panels, corresponding lysates were immunoblotted with anti-Grb2 and antiactin immunoblotting. (C) HEK293 cells were transfected, treated, and stimulated as described for panel B and then processed for immunoprecipitation of pTyr-containing signaling complexes (IP pTyr), followed by anti-Shp2 immunoblotting (top). The middle and bottom panels show anti-Grb2 and antiactin immunoblotting of corresponding lysates. Data in this figure are representative of three independent experiments.