S100A8/A9-induced nuclear factor kappa B (NF-κB) activation and its independence of p38 mitogen-activated protein kinase (MAPK) activation. (a) Activation of the transcriptional factor NF-κB in S100A8/A9-stimulated monocytes. Monocytes (1 × 106 cells per ml in culture medium with 1% fetal calf serum) were stimulated for 30 minutes with or without S100A8/A9 (0.1–10 μM) in the presence of polymyxin B (100 μg/ml). Nuclear proteins were extracted from the cells, and electophoretic mobility shift assay (EMSA) was performed, as described in Materials and methods. Nuclear proteins prepared from monocytes stimulated with 100 ng/ml of lipopolysaccharide were used as positive controls. The specificity of NF-κB protein binding was verified by inhibition and supershift experiments with unlabeled NF-κB consensus or mutant oligonucleotides and anti-NF-κB p65 antibody. (b) Effects of p38 MAPK inhibition on S100A8/A9-induced NF-κB activation. Monocytes, after two-hour pretreatment with or without the MAPK inhibitors (PD98059, SB202190, and SB203580) and the control compound SB202474, were stimulated for 30 minutes with or without S100A8/A9 (10 μM), and nuclear proteins were prepared. The DNA-binding activity of NF-κB was determined by EMSA.