Figure 7. Shp2 knockdown results in defective activation of Ras and MAPKs.

(A) Sensitized RBL cells from the three populations were starved and stimulated with 10 ng/ml DNP for the indicated times. WCL were immunoblotted with anti-p-Erk1/2 and anti-p-JNK and anti-p-p38 antibodies to measure the phosphorylation levels of MAPKs. To control for loading, blots were stripped and reprobed with anti-total Erk1/2, JNK, and p38 antibodies. Shp2 expression was also determined by immunoblotting with anti-Shp2 antibody followed by reprobing with anti-GAPDH antibody. Representative blots of at least 3 experiments are shown. (B) Upper panels, RBL cells from the three populations were sensitized, starved, and stimulated with 10 ng/ml DNP for indicated times and WCL were prepared. Active Ras (Ras-GTP) amounts in WCL were determined by incubating the WCL with GST-RBD, followed by immunoblotting with anti-Ras antibody according to Materials and Methods. 5–10% of WCL of each sample was loaded as a control for the total Ras in RBL cells. Lower panel, activated Ras was measured by quantifying the Ras-GTP levels at 5 min after stimulation normalized to total Ras. Data are expressed as mean ± SEM and representative results from at least three experiments are shown. **, P<0.01, Student t test.