Fig. 1. Abl kinases phosphorylate SHP-2. (A) SHP-2 was immunoprecipitated from 293T cells transfected with SHP-2 and kinase-inactive (KR), wild-type (WT), or constitutively active forms (PP) of Abl or Arg, and blotted with phosphotyrosine antibody (4G10/PY99; top). The blot was stripped and reprobed with SHP-2 antibody (middle). Results are representative of three independent experiments. (B) Immunoprecipitated Abl kinases were incubated in a `cold' kinase assay with GST, GST-SHP-2, or GST-Crk, and probed with anti-phosphotyrosine antibody. Arg-WT was expressed to a greater extent than Arg-PP (data not shown). Data are representative of three independent experiments. (C) Serum-starved 10T1/2-EGFR cells were pretreated with STI571 (10 µM) or vehicle (water) for 4 hours, stimulated with EGF (100 ng/ml), SHP-2 was immunoprecipitated from the lysates, and probed with anti-phosphotyrosine antibody (top). Percentage phosphorylation is relative to total immunoprecipitated SHP-2 protein, and is expressed as a percentage of phosphorylation observed in untreated cells. (D) SHP-2 phosphorylation (relative to total protein levels) from STI571-treated cells (as in C) was compared to untreated cells and expressed as a percentage of untreated. Results from three independent experiments are shown (mean ± s.e.m.). *P0.05 using a ratio paired t-test. (E) 10T1/2-EGFR cells, transfected with Abl or Arg siRNAs (40 nM), were starved and stimulated with EGF for 30 minutes, SHP-2 was immunoprecipitated and blotted with phosphotyrosine antibody. The blot was stripped and reprobed with SHP-2 antibody. SHP-2 phosphorylation (relative to total protein levels) from Abl or Arg siRNA-transfected cells was compared with scrambled control-transfected cells and expressed as a percentage of scrambled control. Since Arg antibodies do not work well for western blots, an in vitro kinase assay was utilized to determine knockdown efficiency (Srinivasan and Plattner, 2006). `Percentage knockdown' is the decrease in protein expression or activity relative to the scrambled control. (F) SHP-2 phosphorylation (relative to total protein levels) from Abl or Arg siRNA-transfected cells (E) was compared with scrambled control-transfected cells and expressed as a percentage of scrambled. Results from three independent experiments are shown; values are mean ± s.e.m. *P0.05, **P<0.005 using a one-way ANOVA followed by a Bonferroni post-hoc test.