FIG. 5.

The Gab1/Shp2 module is required for complete ERK1-2 activation in HEK293 cells, even under high EGF concentrations. (A) HEK293 cells were cotransfected with plasmids encoding His-Myc-tagged ERK1 and Myc-tagged Gab1 constructs (WT or mutated on the Shp2 binding site [Y627F]). After stimulation with EGF (5 or 50 ng/ml) as indicated, cells were lysed and processed for His-tag affinity precipitation, followed by anti-phospho-ERK1-2 immunoblotting. Corresponding lysates were then subjected to anti-Myc immunoblotting to control the expression level of each construct. (B) Same experiment as for panel A, except that cells were cotransfected with plasmids encoding, instead of Gab1 constructs, V5-tagged Shp2 constructs (WT or mutated on the catalytic site [C459G]), as indicated. Associated lysates were then subjected to anti-V5 and anti-Myc immunoblotting. (C) Cells were treated or not treated (−) with siRNA against Gab1, Shp2, or a scrambled sequence (control). Following cell stimulation with EGF (5 or 50 ng/ml) as shown, lysates were subjected to immunoblotting with anti-pERK1-2, -Shp2, -Gab1, and -tubulin antibodies.