FIG. 1.

Reduced activation of Mek and Raf in Shp-2 mutant cells. Fibroblasts of one wild-type (+/+) and two homozygous Shp-2 mutant (−/−) cell lines were starved in serum-free DMEM medium for 36 h and then stimulated with EGF (100 ng/ml) for indicated times. Mek (A) and Raf (B) kinase activities were measured by a coupled in vitro kinase assay as described in the text. Incorporation of ³²P into MBP was determined by scintillation counting to reflect the kinase activities. The protein expression levels of Mek-1 and Raf-1 in two wild-type (+/+), two heterozygous (+/−), and three homozygous mutant (−/−) cell lines were evaluated by immunoblot analysis using specific antibodies against Mek-1 and Raf-1, respectively (C).