Figure 1
Contribution of EphA2-dependent phosphorylation of Shp2 to the growth factor-induced Erk activation. (a) Immunoblot analyses with the antibodies indicated at the left using the cell lysates prepared from the NMuMG cells pretreated with short interfering RNAs (siRNAs) indicated on the top and stimulated with HGF for the time indicated on the top. (b) Quantitative analyses of Erk phosphorylation by HGF in cells treated with control siRNA (control, closed circle) or siRNA against Shp2 (siShp2, open circle). Fold induction after the HGF stimulation was plotted as the value of the intensity of phosphorylated (p)-Erk divided by that of Erk (0 min) was 1. Experiments were performed at least three times. Data are represented as mean with s.d. *P<0.05 between the cells treated with control and those treated with siShp2. (c) Immunoblot analyses with the antibodies indicated at the left using the cell lysates (Total) or the immunoprecipitates with anti-Shp2 (IP: Shp2) prepared from NMuMG cells pretreated with sRNAs indicated on the top and stimulated with HGF for the time indicated on the top. (d) Phosphorylation of Erk observed in (c) was quantified as described in the legend for (b). (e) and (g) Immunoblot analyses with the antibodies indicated at the left using the cell lysates of human umbilical vein endothelial cells (HUVECs) stimulated with fibroblast growth factor-2 (FGF-2) (e) and those of C6 glioma cells stimulated with HGF (g) as described in the legend of (b). (f) and (h) Phosphorylation of Erk observed in (e) and (g) was quantified, respectively, as described in the legend of (b). (i) The number of NMuMG cells treated with either control siRNA (white), EphA2 siRNA (black) or Shp2 siRNA (gray) and cultured in the presence or absence of HGF was counted. The cells transfected for 24 h with siRNAs were replated at the density of 2 × 104 per well in the 12-well plate and were subjected to the calculation at the time after being replated as indicated at the bottom. Data are represented as mean of at least three independent experiments with s.d. *P<0.05 between the cells treated with control and those treated with either EphA2 siRNA or Shp2 siRNA.
