FIGURE 1.

Rac1 is involved in thrombin-induced IL-8/CXCL8 release and IL-8/CXCL8-luciferase activity in A549 cells. A, A549 cells were transiently transfected with either 1 μg of pcDNA (mock) or 0.5 or 1 μg of RacN17 for 24 h. Cells were stimulated with an equivant vehicle control (double-distilled H₂O (−)) or 10 units/ml thrombin for another 24 h, and then IL-8/CXCL8 levels were determined. Data are presented as the mean ± S.E. of three experiments performed in duplicate. *, p < 0.05, as compared with thrombin treatment. B, A549 cells were either transiently transfected with 0.2 μg of IL-8/CXCL8 WT-Luc and 0.5 μg of pBK-CMV-LacZ or cotransfected with 1 μg of pcDNA (mock) or with 0.5 or 1 μg of RacN17 for 24 h and then stimulated with 10 units/ml thrombin for another 24 h. Cells were harvested for the luciferase assay as described under “Experimental Procedures.” Data are presented as the mean ± S.E. of three experiments performed in duplicate. *, p < 0.05, as compared with thrombin treatment. C, for Rac activity, cells were incubated with vehicle or 10 units/ml thrombin for the indicated time intervals, and cell lysates were then immunoprecipitated with PAK-1 PBD-agarose. The Rac activity assay is described under “Experimental Procedures.” Rac1 and α-tubulin protein levels were determined in total lysates by a Western blot analysis as the loading control. Traces represent results from three independent experiments with similar results.