(A,C) 293T cells were transfected with plasmids encoding wild-type or mutant forms of SHP-2 (10 μg), Gab1 (4 μg), and HA-tagged ERK2 (1 μg), cells were serum-starved, stimulated with EGF for the indicated times, and lysates were probed with phospho-ERK1/2 antibody. Only HA-tagged ERK2, which migrates slower than endogenous ERK1/2, is shown. Blots were stripped and reprobed with HA antibody. (B,D) Graphical representations of three unstimulated (A) or four EGF-stimulation experiments (C). Levels of phosphorylated HA-ERK2 relative to total HA-ERK2 for cells expressing SHP-2 mutants were expressed as a percentage of HA-ERK2 phosphorylation observed in cells expressing wild-type SHP-2. Results are mean ±s.e.m. *p≤0.05, **p≤0.03, ***p≤0.001 using t-tests.