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. 2005 Sep 26;391(Pt 1):143–151. doi: 10.1042/BJ20050229

Figure 2. Effects of SHP2-binding defective Gab1 and Gab2 mutants on ERK activation.

Figure 2

(A) T47D cells in 60 mm plates were co-transfected with HA–ERK2 (0.4 μg) and Gab1FF, Gab2F or an empty vector (3.6 μg). Transfected cells were serum-starved and stimulated with EGF (5 ng/ml, 5 min) or mock-treated. An aliquot of each cell lysate supernatant (10 μg) was analysed for expression of Gab1FF and Gab2F by immunoblotting with an anti-FLAG antibody (lower panel). HA–ERK2 kinase activity was determined by an immunocomplex kinase assay using MBP as substrate. Reaction mixtures were separated by SDS/polyacrylamide gel and transferred on to a nitrocellulose membrane. After detection of MBP phosphorylation, the membrane was used for immunoblot analysis to determine the relative amount of HA–ERK2 protein in each sample. (B) COS-7 cells in 60 mm plates were co-transfected with HA–ERK2 (0.2 μg) and Gab1FF, Gab2F or an empty vector (1.8 μg) as indicated. Transfected cells were serum-starved and stimulated with EGF (0.5 ng/ml, 5 min) or mock-treated. Gab1FF and Gab2F expression was examined and ERK2 kinase activities were determined as above. Graphs represent averages and ranges of the ERK2 kinase activity from two independent experiments.