Figure 4. Inhibition of EGF-stimulated Mek1 activation in T47D and MCF-7 cells by Gab1siRNA and Gab2siRNA.

(A, D) T47D cells or MCF-7 cells in 60 mm plates were co-transfected with HA–Mek1 (0.4 μg) and pGab1siRNA, pGab2siRNA, pGab1/pGab2siRNA, or the empty vector (3.6 μg). Cells were serum-starved for 20 h after 24 h transfection, and then stimulated with EGF (5 ng/ml, 5 min) or mock-treated with BSA. HA–Mek1 was immunoprecipitated and Mek1 kinase activity was determined by phosphorylation of a kinase-defective ERK2 (KR) in the presence of [γ-³²P]ATP. After autoradiography and phosphoimaging analysis, membranes were subjected to immunoblotting analysis of HA–Mek1. The histogram in (A) represents the averages and ranges from two independent experiments. (B, C) T47D cells in 60 mm plates were transfected with HA–Mek1 (0.4 μg)+siRNA and mouse Gab expression plasmids (1.8 μg/each) or control vector (−) as indicated. EGF-induced HA–Mek1 activation was measured as in (A and D).