Degradation of inhibitor of kappaB-alpha (I-κB-α) in RANK-L-activated neutrophils. I-κB-α was detected in whole-cell lysates by Western blotting as described in Materials and methods. Freshly isolated neutrophils (a), or blood neutrophils pretreated for a 3-day incubation period in rheumatoid arthritis-synovial fluid (RA-SF) (80%) + control medium (20%) (b), were stimulated with 50 ng/ml tumor necrosis factor-alpha (TNF-α) for 10 minutes or with 100 ng/ml RANK-L for 10 and 20 minutes. Western blotting was performed with a rabbit anti-human I-κB-α antibody, a horseradish peroxidase-conjugated anti-rabbit immunoglobulin G antibody, and the enhanced chemiluminescence detection system. Results shown are representative of neutrophils from three different healthy subjects incubated in three different RA-SF. RANK-L, ligand of receptor activator of nuclear factor-kappa-B.