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Fig. 2. Specific association between Grb2 and Sprouty1 or Sos1 and Sprouty4. (A) 293T cells were transfected with the expression plasmid encoding either Flag-tagged Sprouty1, Sprouty2, Sprouty3 or Sprouty4 (0.5 µg). After 24 hours, cells were serum-starved for 6 hours and then mock-treated (C) or treated with 20 ng/ml EGF (E) or 20 ng/ml FGF-2 (F) for 15 minutes. The cell lysates (500 µg protein) were subjected to immunoprecipitation (IP) using anti-Grb2 antibody, anti-Sos1 antibody, or anti-RasGAP antibody, followed by immunoblotting with anti-Flag antibody (for Sprouty proteins), anti-Grb2 antibody, anti-Sos1 antibody or anti-RasGAP antibody. Rabbit non-immune IgG was used as a control. Total cell lysates (50 µg protein) were subjected to immunoblot analysis with anti-Flag antibody to show the expression levels of Sprouty proteins. (B) 293T cells transfected with the expression plasmid encoding Myc-tagged Sprouty1 or Sprouty2 were treated with 20 ng/ml EGF for 15 minutes. Cell lysates (500 µg protein) were subjected to a pull-down assay using Grb2-agarose beads (Grb2) or glutathione-sepharose 4B beads (Glut), followed by immunoblotting with anti-Myc antibody (for Sprouty1/2). Total cell lysates (50 µg protein) were subjected to immunoblot analysis with anti-Myc antibody to show the expression levels of exogenous Sprouty1/2 (Lys). (C) 293T cells were co-transfected with expression plasmids encoding Myc-tagged Sprouty1 and each of either Flag-tagged Sprouty2, Sprouty3 or Sprouty4, or co-transfected with expression plasmids encoding Myc-tagged Sprouty4 and each of either Flag-tagged Sprouty1, Sprouty2 or Sprouty3 as indicated (0.5 µg each). After 24 hours, the cells were serum-starved for 6 hours and then mock-treated (C) or treated with 20 ng/ml EGF (E) or 20 ng/ml FGF-2 (F) for 15 minutes. Cell lysates (500 µg protein) were subjected to immunoprecipitation using anti-Grb2 antibody or anti-Sos1 antibody, followed by immunoblotting with anti-Flag antibody, anti-Myc antibody, anti-Grb2 antibody, or anti-Sos1 antibody. Similar results were obtained in three independent experiments.





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