FIGURE 3.

Ang1 induces Dll4 expression through AKT-mediated inhibition of GSK3β. A, confluent HUVECs starved for 12 h were stimulated with COMP-Ang1 for the periods indicated at the top (h). Cell lysates were subjected to Western blot analysis with anti-phospho-GSK3β (P-GSK3β), anti-GSK3β (GSK3β), and anti-tubulin (tubulin) antibodies. B, 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 30 min. Cell lysates were subjected to Western blot analysis with anti-phospho-GSK3β (P-GSK3β), anti-GSK3β (GSK3β), anti-phospho-AKT (P-AKT), and anti-AKT (AKT) antibodies. C, phosphorylated GSK3β levels observed in B are quantified by normalizing the expression of phosphorylated GSK3β by that of total GSK3β. Values are expressed as fold-induction relative to that in the wortmannin-untreated cells stimulated with vehicle, and shown as mean ± S.D. of three independent experiments. D, confluent HUVECs were infected with adenoviruses encoding either LacZ or CA-AKT. Cell lysates were subjected to Western blot analysis as described in B. E, confluent and sparse HUVECs were starved in medium 199 containing 1% BSA for 12 h, and treated with 10 μm SB216763 for the periods indicated at the top (h). Cell lysates were subjected to Western blot analysis with anti-Dll4 (Dll4), anti-NICD (NICD), and anti-tubulin (tubulin) antibodies. F, the effect of 20 mm LiCl on the expression of Dll4 and NICD was analyzed as described in E. In C, a significant difference between two groups is indicated as: *, p < 0.05. n.s. indicates no significance between two groups.