Fig. 4. Co-expression of Sprouty1 and Sprouty4 efficiently inhibits the FGF-2-induced association of Grb2 with FRS2. 293T cells were co-transfected with expression plasmids encoding HA-tagged Grb2 (0.5 µg) and vector control (–), Flag-tagged Sprouty1 (1 µg), Flag-tagged Sprouty4 (1 µg), or Flag-tagged Sprouty1 and Sprouty4 in combination (0.5 µg each) as indicated. After 24 hours, cells were serum-starved for 6 hours and then mock-treated (Control) or treated with 20 ng/ml EGF or 20 ng/ml FGF-2 for 15 minutes. Cell lysates (500 µg protein) were subjected to immunoprecipitation (IP) using anti-HA antibody, followed by immunoblotting with anti-FRS2 antibody, anti-Shc antibody, anti-Grb2 antibody or anti-Flag antibody (for Sprouty1/4). The relative intensity of FRS2 band, as compared with that of the respective Grb2 signal, was determined by using the Multi Gauge software, and normalized to 1.00 for control cells without exogenous Sprouty protein(s) (FRS2/Grb2). Total cell lysates (50 µg protein) were subjected to immunoblot analysis with anti-Flag antibody to show the expression levels of exogenous Sprouty1/4. Similar results were obtained in three independent experiments.