Figure 3.

Stable overexpression of HA-tagged constitutively active P110 Pi(3)K-CAAX in NIH 3T3 cells results in increased ILK activity and constitutive phosphorylation of PKB/AKT on serine-473. NIH 3T3 cell clones overexpressing constitutively active membrane-targeted p110 subunit of Pi(3)K, or kinase-deficient P110 (15), were serum-starved for 18 hr and then analyzed for ILK activity by using MBP, or for PKB/AKT serine-473 phosphorylation. Pi(3)K activity was inhibited by preincubating the cells with Ly294002 (50 μM) for 15 min. P110 expression was detected in the transfectants by Western blotting with anti-HA antibody (data not shown).