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Figure 3


Fig. 3. Endogenous Abl kinases phosphorylate SHP-2 on Y580, and are required for sustained ERK activation in response to PDGF. (A) Serum-starved 10T1/2-EGFR cells were pretreated with STI571 (10 µM), stimulated with EGF, and lysates were probed with phospho-specific SHP-2 antibodies (Y580, Y542). Percentage phosphorylation is relative to total SHP-2 protein levels obtained from reprobed blots. (B) Percentage phosphorylation in lysates probed with phospho-SHP-2-Y580 antibody (as in A). Values are mean ± s.e.m. of three independent experiments. *P≤0.05 using a ratio paired t-test. (C) 10T1/2-EGFR or NIH3T3 cells, transfected with Abl or Arg siRNAs (40 nM), were serum-starved, stimulated with EGF (100 ng/ml; 10T1/2-EGFR) or PDGF-BB (12.5 ng/ml; NIH3T3) for 30 or 10 minutes, respectively, and lysates were probed with the indicated antibodies. SHP-2-Y580 phosphorylation in Abl or Arg siRNA-transfected cells was expressed as a percentage of SHP-2-Y580 phosphorylation observed in scrambled control-transfected cells and is relative to total SHP-2 protein levels obtained from reprobed blots. (D) Results from three independent experiments as in C. Values are mean ± s.e.m.*P≤0.05, ***P≤0.005 using a one-way ANOVA followed by a Bonferroni post-hoc test. (E) NIH3T3 cells, transfected with siRNAs, were serum-starved, stimulated with PDGF-BB (12.5 ng/ml) for 30 minutes, and phosphorylation of ERK1/2 was assessed by western blotting with a phospho-specific antibody [T(P)EY(P)]. ERK phosphorylation shown is relative to total ERK protein levels obtained from reprobed blots, and is expressed as a percentage of phosphorylation observed in scrambled control-transfected cells. (F) Results from three independent experiments as in E. Values are mean ± s.e.m.*P≤0.05, **P≤0.005, ***P≤0.001 using a one-way ANOVA followed by a Bonferroni post-hoc test.