Fig. 6. siRNA knockdown of Sprouty1/Sprouty4 induces a prolonged activation of ERK1/2 in FGF-2-stimulated Swiss 3T3 cells. (A) Swiss 3T3 cells were transfected with Sprouty1 siRNA (1), Sprouty4 siRNA (4), Sprouty1 siRNA and Sprouty4 siRNA in combination (1+4), or matched scrambled RNAs to Sprouty1 siRNA and Sprouty4 siRNA in combination (C). After 24 hours, cells were serum-starved for 24 hours and then stimulated with FGF-2 (20 ng/ml) for 1 hour (for the analysis of Sprouty1 expression) or 2 hours (for the analysis of Sprouty4 expression). Total RNA was isolated and RT-PCR (27 cycles) was performed for Sprouty1 or Sprouty4 to generate 372 bp fragment or 443 bp fragment, respectively. A portion of GAPDH was co-amplified as an internal control (250 bp). The relative intensity of Sprouty1/4 band, as compared with that of the respective GAPDH signal, was determined by using the Multi Gauge software, and normalized to 1.00 for the respective scramble RNA-transfected control cells stimulated with FGF-2 for 0.5 hours. M, DNA ladder markers. (B) Swiss 3T3 cells were transfected with Sprouty1 siRNA (Sprouty1), Sprouty4 siRNA (Sprouty4), Sprouty1 siRNA and Sprouty4 siRNA in combination (Sprouty1 + Sprouty4), or matched scrambled RNAs to Sprouty1 siRNA and Sprouty4 siRNA in combination (Control). After 24 hours, cells were serum-starved for 24 hours and then stimulated with FGF-2 (20 ng/ml) for the indicated periods of time. Total cell lysates (10 µg protein) were subjected to immunoblot analysis with anti-ppERK1/2 antibody or anti-ERK1/2 antibody. The relative intensity of phosphorylated ERK1 and ERK2 bands, as compared with that of the respective ERK1 and ERK2 signals, was determined by using the Multi Gauge software, and normalized to 1.00 for the respective Swiss 3T3 cells stimulated with FGF-2 for 1 hour (ppERKs/ERKs). Similar results were obtained in two independent experiments.