FIGURE 2.

Ang1 induces Dll4 expression through a PI3K/AKT pathway. A, confluent HUVECs starved for 12 h were pretreated with vehicle (control) or 60 nm wortmannin for 30 min, and subsequently stimulated with vehicle (−) or COMP-Ang1 (+) for 1 h. Dll4 protein expression was examined by Western blot analysis as described in the legend of Fig. 1C. B, expression of Dll4 protein observed in A are quantified as described in legend of Fig. 1D. Values are expressed as fold-induction relative to that in the wortmannin-untreated cells stimulated with vehicle, and shown as mean ± S.D. of five independent experiments. C, confluent HUVECs starved for 12 h were pretreated with vehicle (control) or 8 μm AKT inhibitor for 10 min and subsequently stimulated with vehicle (−) or COMP-Ang1 (+) for 1 h. Dll4 protein expression was analyzed as described in A. D, expression of Dll4 protein observed in C are quantified as described in legend of Fig. 1D. Values are expressed as fold-induction relative to that in the AKT inhibitor-untreated cells stimulated with vehicle, and shown as mean ± S.D. of four independent experiments. E, confluent HUVECs were infected with adenoviruses encoding LacZ or with two different titers of adenoviruses encoding AKT-CA for 48 h. Cell lysates were subjected Western blot analysis with anti-Dll4 (Dll4), anti-NICD (NICD), anti-phospho-AKT (P-AKT), anti-AKT (AKT), and anti-tubulin (tubulin) antibodies. Significant differences between two groups (B and D) are indicated as: **, p < 0.01. n.s. indicates no significance between two groups.