The EMSA was performed essentially as described previously [24]. Protein extracts (10 µg) were added to the binding mixture (20 µl of 10 mM Tris-HCl, pH 7.5, 20 mM KCl, 1 mM EDTA, 7.5% glycerol, 1.5 mM DTT, 20 µg BSA and 40–60,000 cpm [32P] –labeled oligonucleotide) in duplicates and incubated for 20–30 minutes at room temperature. The reaction mixture was brought to 0.6% or 0.03% deoxycholate (DOC) after 30 min followed by the addition of 1.2% NP-40 and were loaded onto a 5% polyacrylamide gel in 0.5×TGE (25 mM Tris-HCl, 0.19 M glycine, 1 mM EDTA, pH 8.5) buffer. Reference samples consisting of pooled cerebellar extracts from control subjects were loaded onto two wells on each edge of the gel to ascertain reproducibility of the gel, and also for inter-gel comparison. In competition experiments, 10 ng of unlabeled wt-κB or m-κB oligonucleotide was added to the incubation mixture prior to the initiation of the binding reaction. After electrophoresis the gels were fixed in 40% methanol containing 3% acetic acid for 10 min, dried and exposed to intensifying screen. Detection was performed on phosphorimager BAS 1500 (Fuji Film, Kanagawa, Japan) and densities were analyzed using Fuji Film Image Gauge software.
