Powdered tissue samples were boiled in pre-warmed SDS extraction buffer (0.45 M Tris-HCl, pH 8.5, 2.5% glycerol, 4% SDS, 0.5 mM DTT and 5×protease inhibitors for 5 min, sonicated and protein extracts were aliquoted and kept at −80°C until usage. Protein concentration was determined with the DC protein assay (Bio-Rad Laboratories, Hercules, CA, USA). Aliquots of tissue extracts (50–120 µg protein) were heated for 5 min at 95°C in the presence of 5 mM β-mercaptoethanol and resolved by SDS-PAGE on 10% Tricine gels as described earlier [37]. Reference samples consisting of pooled cerebellar extracts from control subjects were loaded onto three wells, on each edge and in the middle of each gel and their density values were used to ascertain reproducibility on each blot and interblot comparison. Proteins were transferred onto nitrocellulose membranes (Schleicher and Schuell, Dassel, Germany) at 4°C and stained with Memcode Reversible Protein Stain Kit (Pierce, Rockford, USA). Densitometry values of protein load were used for normalization of Western blot data. After destaining, membranes were incubated with stripping buffer (62.5 mM Tris-HCl, 2% SDS, 100 mM β-mercaptoethanol,
