FIGURE 7.

Involvement of Rac1, PI3K, and Akt in thrombin-induced IKKα/β kinase activity in A549 cells. A–C, cells were transfected with1 μg of pcDNA (mock), 1 μg of RacN17 (A), or 0.5 μg of AktDN (C) for 24 h or were pretreated with an equivalent vehicle control (DMSO) or 10 μm LY294002 for 30 min (B) and then incubated with vehicle or 10 units/ml thrombin for another 30 min. Whole-cell lysates were then immunoprecipitated with antibodies specific for IKKα and IKKβ. One set of immunoprecipitates was subjected to the kinase assay (KA) described under “Experimental Procedures” using the IκBα-GST fusion protein as a substrate. The other set of immunoprecipitates was subjected to 10% SDS-PAGE and analyzed by immunoblotting (IB) with the anti-IKKα/β antibody. Equal amounts of the immunoprecipitated kinase complex present in each kinase assay were confirmed by immunoblotting for IKKα/β. α-Tubulin protein in total cell lysates served as the loading control. Typical traces represent two experiments with similar results.