FIGURE 5.

Involvement of PI3K in thrombin-induced Akt Ser-473 phosphorylation and kinase activity in A549 cells. A and C, cells were pretreated with an equivalent vehicle control (DMSO), 10 μm LY294002 (A), or 3 μm Ro-32-0432 (C) for 30 min and then incubated with the vehicle or 10 units/ml thrombin for another 30 min. Akt Ser-473 phosphorylation was shown by immunoblotting with an antibody specific for phosphorylated Akt Ser-473. Equal loading in each lane is shown by the similar intensities of Akt1/2 or α-tubulin. Typical traces represent three experiments with similar results. Results are expressed as the mean ± S.E. (n = 3). *, p < 0.05, as compared with the thrombin-treated group. B, cells were pretreated with an equivalent vehicle control (DMSO) or 10 μm LY294002 for 30 min and then incubated with vehicle or 10 units/ml thrombin for another 30 min. Cell lysates were then immunoprecipitated with antibodies specific for Akt1/2. One set of immunoprecipitates was subjected to the kinase assay (KA) described as in the legend for Fig. 4. The other set of immunoprecipitates was subjected to 10% SDS-PAGE and analyzed by immunoblotting (IB) with the anti-Akt1/2 antibody. Equal amounts of the immunoprecipitated kinase complex present in each kinase assay were confirmed by immunoblotting for Akt1/2. α-Tubulin protein in total cell lysates served as the loading control. Typical traces represent two experiments with similar results.