Fig. 4. SHP-2 phosphorylation mutants have altered activities towards phosphotyrosine-containing substrates. (A) Wild-type or mutant forms of SHP-2 were immunoprecipitated from serum-starved, overexpressing 293T cells, incubated with pNPP substrate and supernatant absorbance at 405 nm (A₄₀₅) was measured. Some error bars are too small to be visible. (B) Wild-type or mutant forms of SHP-2 were immunoprecipitated from serum-starved overexpressing 293T cells, incubated with 250 µM substrate [R-R-L-I-E-D-A-E-Y(P)-A-A-R-G] for 30 minutes at 37°C, and phosphate release into the supernatant was quantified by incubation with Malachite Green, and measurement of A₆₂₀. Absorbance values were compared to a standard phosphate curve to determine pmoles of phosphate released. Values obtained for mutant forms of SHP-2 were expressed as a percentage of wild-type; values are mean ± s.e.m. from three independent experiments. ***P0.001 using one-way ANOVAs followed by Bonferroni post-hoc tests. Immunoprecipitates were blotted with SHP-2 antibody (representative experiments are shown). (C) SHP-2 immunoprecipitates from expressing 293T cells (as in B) were incubated in a phosphatase assay with increasing concentrations of peptide substrate. Velocity values were converted to pmol/minute/pmol of enzyme utilized. A representative experiment is shown. (D) Velocity values from two independent experiments (as in C) were standardized by converting the values to percentage of the maximum obtained in each experiment; values are mean ± s.e.m. (some error bars are too small to be visible).