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Fig. 3. Co-expression of Sprouty1 and Sprouty4 efficiently suppresses ERK activation induced by FGF-2. (A) 293T cells were co-transfected with expression plasmids encoding HA-tagged ERK2 (0.5 µg) and each of either vector control (–), Flag-tagged Sprouty1, Sprouty2 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, 20 ng/ml FGF-2 or 10 ng/ml PMA for 15 minutes. Cell lysates (500 µg protein) were subjected to immunoprecipitation (IP) using anti-HA antibody, followed by immunoblotting (IB) with anti-ppERK1/2 antibody. An anti-HA blot demonstrates equal amounts of immunoprecipitated ERK2. Total cell lysates (50 µg protein) were subjected to immunoblot analysis with anti-Flag antibody to show the expression levels of exogenous Sprouty1/2/4. (B) 293T cells were co-transfected with expression plasmids encoding HA-tagged ERK-2 (0.5 µg) and vector control (–), Flag-tagged Sprouty1 (1 µg), Flag-tagged Sprouty2 (1 µg), Flag-tagged Sprouty4 (1 µg), Flag-tagged Sprouty1 and Sprouty2 in combination (0.5 µg each), Flag-tagged Sprouty1 and Sprouty4 in combination (0.5 µg each), or Flag-tagged Sprouty2 and Sprouty4 in combination (0.5 µg each) as indicated. ERK activation induced by FGF-2 or EGF was analyzed as described above. The relative intensity of phosphorylated HA-ERK2 band, as compared with that of the respective HA-ERK2 signal, was determined by using the Multi Gauge software, version 3.0 (Fuji Photo Film, Tokyo), and normalized to 1.00 for control cells without exogenous Sprouty protein(s) (ppERK2/ERK2). Total cell lysates (50 µg protein) were subjected to immunoblot analysis with anti-Flag antibody to show the expression levels of exogenous Sprouty1/2/4. Similar results were obtained in three independent experiments.
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