FIGURE 4.

Involvement of Rac1 in thrombin-mediated Akt Ser-473 phosphorylation and kinase activity in A549 cells. A, cells were transfected with 1 μg of pcDNA (mock) or 1 μg of RacN17 for 24 h 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 transfected with 1 μg of pcDNA (mock) or 1 μg of RacN17 for 24 h 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 under “Experimental Procedures” using the histone H2B as a substrate). 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.