FIG. 10.

Rescue of defective Erk induction by EGF in Shp-2^−/− cells and RT-PCR analysis of the fsp-1 mRNA expression. (A) Vector-transfected control cell line (Con.) and two cell clones expressing different levels of wild-type Shp-2 were stimulated with EGF (100 ng/ml) for indicated times. Erk protein was immunoprecipitated from cell lysates, and the kinase activity was measured by the in vitro kinase assay as described previously (45). (B) Total RNA of different cell lines was purified from 3 × 10⁶ cells by using an RNeasy Mini kit from Qiagen as specified by the manufacturer. Gene-specific primers for mouse fsp-1 (sense primer, 5′-CAG CGA AAG AGG GTG ACA AGT TCA-3′; antisense primer, 5′-ATG TGC GAA GAA GCC AGA GTA AGG-3′) (46, 49) or hypoxanthine phosphoribosyltransferase (HPRT), as a positive control (38), were used to amplify 1 μg of RNA, using a GeneAmp RNA PCR kit (Perkin-Elmer) as instructed by the manufacturer. RT-PCR products were resolved in 1.2% agarose gel and visualized by ethidium bromide staining.