Fig. 4. Gab1 overexpression significantly enhances the mitogenic response of 10W+8 to EGF stimulation. A, Gab1 was immunoprecipitated (IP) from the lysates of parental 10W+8 (Lane 1), 10W+8-mock (Lane 2), and 10W+8-Gab1 (Lane 3). The samples were analyzed by SDS-PAGE (8%), followed by anti-Gab1 blotting and detection with the ECL system. Gab1 expression in 10W+8-Gab1 was approximately 2-fold of that in 10W+8 or 10W+8-mock. B, 10 µg of total RNA from 10W+8 (Lane 1), 10W+8-mock (Lane 2), and 10W+8-Gab1 (Lane 3) were examined for the expression of Gab1 mRNA. 10W+8-Gab1 shows exogenous expression of mRNA in addition to endogenous 5.2-kb mRNA (top panel, Lane 3). Equal loading of total RNA is visualized under UV light with an ethidium bromide staining of the gel (bottom panel). C, 10W+8-mock and 10W+8-Gab1 were serum-starved for 16 h and incubated with 1 µCi/well [3H]thymidine in the presence or absence of 50 ng/ml EGF at 37°C for 24 h. The radioactivity of the incorporated [3H]thymidine was measured. Gab1 overexpression resulted in a 2-fold increase in the mitogenic activity of 10W+8 in response to EGF stimulation. The data are the means ± SD of six determinations. *, P < 0.05 versus 10W+8-mock, EGF (+).