Specificity of C5a-induced κB binding activity. Nuclear extracts prepared from unstimulated PBMC (basal; lane 1), PBMC stimulated with TNF (40 ng/mL for 2 hours; lane 2), and C5a (200 nmol/L for 2 hours; lanes 3 through 9) were used for analysis of κB binding activity by EMSA, in the absence (lane 3) or presence (lanes 4 and 5) of competitive unlabeled (cold) oligonucleotide probes. Lane 4 contains an oligonucleotide with the κB consensus sequence at 100-fold molar excess; lane 5 has an olinonucleotide with AP-1 consensus sequence at 100-fold molar excess. The κB DNA-protein complexes are marked with brackets. Lanes 6 through 9, nuclear extracts from cells stimulated with C5a were incubated in the presence of Ab (2 μg/sample) against NF-κB/Rel proteins: lane 6, anti-p50; lane 7, anti-p65; and lane 8, anti-p50 plus anti-p65. Lane 9 contains anti-p50 and anti-p65 plus the neutralizing peptides used for antibody production. Samples were analyzed by EMSA, using 6% acrylamide gel and³²P-labeled double-stranded oligonucleotide containing the NF-κB binding site consensus sequence. The gel supershift bands induced by the anti-p50 and anti-p65 antibodies were indicated with arrows.