C5a-induced transcription factor activation in PBMC. For each sample, 5 × 10⁶ freshly prepared PBMC were incubated with 200 nmol/L of human C5a at 37°C for different lengths of time (0 to 4 hours). The cells were then harvested and nuclear extracts were prepared as described in Materials and Methods. Nuclear extract (2.5 μg) from each time point was incubated with one of the three different probes: ³²P-labeled, double-stranded oligonucleotides containing binding sites for κB, AP-1, and NF-IL-6. The binding mixture was subjected to 5% acrylamide gel electrophoresis. The binding activities for NF-κB (top panel), AP-1 (middle panel), or NF-IL-6 (lower panel) were measured by EMSA. The DNA-protein complexes are marked with brackets. TNF (40 ng/mL)-stimulated cells were used as positive controls for NF-κB and AP-1 activation, and LPS (5 μg/mL; lane 6) was used as a positive control for NF-IL-6 binding activity.