text: Gel mobility-shift assay of NF-kappaB binding site
  Nuclear proteins were extracted from about 5 x 106 PBMC. Oligonucleotide probes encompassing the NF-kappaB 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 [gamma-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 mug of protein) were incubated with radiolabeled DNA probes (10 ng; 100,000 c.p.m.) for 30 min at room temperature in 20 mul 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/mul poly(dI-dC). Samples were subjected to electrophoresis on nondenaturing 5% polyacrylamide gels in 0.5 x Tris-borate-EDTA buffer (pH 8.0) at 100 V. Gels were dried under vacuum and exposed to Kodak X-OMAT film at -70degreesC with intensifying screens. Rabbit polyclonal antibodies against NF-kappaB subunits p50, p65 and c-Rel were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). 
  