Figure 7
Figure 7. Impaired signaling in Rap1b−/− lung endothelial cells. (A) Typical experiment showing time course of Rap1 activation in normal (WT) or Rap1b-deficient (KO) cells in response to treatment with VEGF. GTP-bound Rap1 was pulled down with RalGDS-GST and detected by an anti-Rap1 polyclonal antibody that recognizes both Rap1 isoforms (top blot). The graph depicts quantitation of fold induction of Rap1 activation by VEGF in WT cells in 5 experiments. The values were obtained by normalizing Rap1 signal to actin content in a corresponding lysate sample (bottom blot). Fold induction of Rap1-GTP loading was calculated by dividing values normalized for actin content from VEGF-treated samples by values from nontreated control samples. Fold induction values obtained from 5 experiments were averaged and are expressed as a percentage of nontreated controls; error bars are SEM. (B,C) Decreased MAPK activation in Rap1b-deficient lung endothelial cells. Western blot analysis of the phosphorylation level of p38 MAPK (B) and ERK (C) in normal (WT) or Rap1b-deficient (KO) endothelial cells cultured and serum-starved for 4 hours (SF) or serum-starved and stimulated for 10 minutes with 50 ng/mL of the indicated growth factor (top blots). Shown are representative experiments. The graphs depict quantitation of fold induction of MAPK phosphorylation by the indicated growth factor from 3 separate experiments. To normalize for protein content, the blots were stripped and reprobed with actin-specific antibody (bottom blots). Fold induction of MAPK phosphorylation was calculated by dividing values from growth factor–treated samples normalized for actin content by values from serum-free control samples. The values for fold induction of MAPK phosphorylation of knockout samples (▧) were expressed as percentage of wild-type values (■). Such derived values from individual experiments were averaged and mean fold induction was plotted with SEM as error bars. Bars represent fold induction of MAPK, which was calculated by dividing values from VEGF-treated samples normalized for actin content by values from nontreated control samples.

Impaired signaling in Rap1b−/− lung endothelial cells. (A) Typical experiment showing time course of Rap1 activation in normal (WT) or Rap1b-deficient (KO) cells in response to treatment with VEGF. GTP-bound Rap1 was pulled down with RalGDS-GST and detected by an anti-Rap1 polyclonal antibody that recognizes both Rap1 isoforms (top blot). The graph depicts quantitation of fold induction of Rap1 activation by VEGF in WT cells in 5 experiments. The values were obtained by normalizing Rap1 signal to actin content in a corresponding lysate sample (bottom blot). Fold induction of Rap1-GTP loading was calculated by dividing values normalized for actin content from VEGF-treated samples by values from nontreated control samples. Fold induction values obtained from 5 experiments were averaged and are expressed as a percentage of nontreated controls; error bars are SEM. (B,C) Decreased MAPK activation in Rap1b-deficient lung endothelial cells. Western blot analysis of the phosphorylation level of p38 MAPK (B) and ERK (C) in normal (WT) or Rap1b-deficient (KO) endothelial cells cultured and serum-starved for 4 hours (SF) or serum-starved and stimulated for 10 minutes with 50 ng/mL of the indicated growth factor (top blots). Shown are representative experiments. The graphs depict quantitation of fold induction of MAPK phosphorylation by the indicated growth factor from 3 separate experiments. To normalize for protein content, the blots were stripped and reprobed with actin-specific antibody (bottom blots). Fold induction of MAPK phosphorylation was calculated by dividing values from growth factor–treated samples normalized for actin content by values from serum-free control samples. The values for fold induction of MAPK phosphorylation of knockout samples (▧) were expressed as percentage of wild-type values (■). Such derived values from individual experiments were averaged and mean fold induction was plotted with SEM as error bars. Bars represent fold induction of MAPK, which was calculated by dividing values from VEGF-treated samples normalized for actin content by values from nontreated control samples.

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