Gel mobility-shift assay of NF-κB binding site
Nuclear proteins were extracted from about 5 × 106 PBMC. Oligonucleotide probes encompassing the NF-κB binding site of the human IL-17 promoter (5'-ATG ACC TGG AAA TAC CCA AAA TTC-3') were generated by 5'-end labeling of the sense strand with [γ-32P]dATP (Amersham Pharmacia) and T4 polynucleotide kinase (TaKaRa). Unincorporated nucleotides were removed by NucTrap probe purification columns (Stratagene, La Jolla, CA, USA). Nuclear extracts (2 μg of protein) were incubated with radiolabeled DNA probes (10 ng; 100,000 c.p.m.) for 30 min at room temperature in 20 μl of binding buffer consisting of 20 mM Tris-HCl, pH 7.9, 50 mM KCl, 1 mM dithiothreitol, 0.5 mM EDTA, 5% glycerol, 1 mg/ml BSA, 0.2% Nonidet P40 and 50 ng/μl poly(dI-dC). Samples were subjected to electrophoresis on nondenaturing 5% polyacrylamide gels in 0.5 × Tris-borate-EDTA buffer (pH 8.0) at 100 V. Gels were dried under vacuum and exposed to Kodak X-OMAT film at -70°C with intensifying screens. Rabbit polyclonal antibodies against NF-κB subunits p50, p65 and c-Rel were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).