Correlation between C5a-induced κB binding activity and IL-8 gene transcription. The murine RAW264.7 macrophage cells were transfected with 8 μg plasmid DNA for either the IκB promoter-CAT reporters (lanes 1 through 4) or the p-272CAT reporters (lanes 5 through 8), together with 2 μg of the control plasmid pCMVβ DNA to monitor transfection efficiency. Relative CAT activities were determined in these cells after 2 hours of stimulation with C5a by normalizing the CAT activities to the β-galactosidase activities. The acetylated forms of CAT (AcCM) are marked with brackets. The net fold induction of relative CAT activities by C5a is shown at the bottom. Lane 1, no reporter plasmid was transfected. The p0.2kb(WT)CAT plasmid was used in the absence (lane 2) or presence (lane 3) of human C5a. Lane 4 was a sample with p0.2kb(M)CAT, which contains a mutated κB site in the IκB promoter. In lanes 5 through 8, CAT reporter gene plasmids driven by the IL-8 promoter were used in the absence (lanes 5 and 7) or the presence (lanes 6 and 8) of C5a. Lanes 5 and 6 contain the p-272CAT construct. A reporter with the κB binding sequence removed p-94(Δ 78-71) CAT was used in lanes 7 and 8. The thin-layer chromatography data shown here is representative of three CAT assays with similar results.