Fig. 5. Hetero-oligomer formation of Sprouty1/2 and Sprouty4 in FGF-2-stimulated Swiss 3T3 cells. (A) Swiss 3T3 cells were serum-starved for 24 hours and then stimulated with 20 ng/ml of FGF-2 for the indicated periods of time. In some experiments, the cells were pretreated with 10 µM PD184352 (PD) for 30 minutes and then stimulated with FGF-2. Total cell lysates (50 µg protein) were subjected to immunoblot analysis with anti-Sprouty1 antibody, anti-Sprouty2 antibody, anti-Sprouty4 antibody, anti-ppERK1/2 antibody, anti-ERK1/2 antibody, anti-Sos1 antibody or anti-Grb2 antibody. (B) Swiss 3T3 cells were serum-starved for 24 hours and then stimulated with FGF-2 (20 ng/ml) for the indicated periods of time. Cell lysates (500 µg protein) were then subjected to immunoprecipitation (IP) using the anti-Sprouty4 antibody (2 µg each of sc-18607 and sc-18609), following by immunoblotting with anti-Sprouty1 antibody, anti-Sprouty2 antibody, anti-Sprouty4 antibody (sc-18607), anti-Sos1 antibody, or anti-Grb2 antibody. Similar results were obtained in three independent experiments.