To determine downstream effector molecules of the PI3K pathway, we evaluated the activation of Akt by western blotting. As shown in Fig. 6, at 10 min of incubation with anti-CD3 (10 μg/ml) or LY294002 (20 μM), no difference in the amounts of phosphorylated Akt was observed. However, after 30 min of incubation, phosphorylated Akt increased (lane 2), and the effect of inhibition by LY294002 (lane 3) reached a peak at 60 min, lasting to 120–240 min. In contrast, non-phosphorylated Akt and β-actin remained unchanged regardless of incubation time. PHA, concanavalin A and IL-15 also demonstrated the same effect on phosphorylated Akt as shown with anti-CD3, which was an inhibition by wortmannin and PDTC as well as by LY294002 (data not shown).