Immunoblot assay

For protein preparation, brain tissue was boiled at 95 °C for 5 min in 2× lysis buffer (Laemmli 1970) and cooled in an ice bath for 5 min. Particulate matter was removed by centrifuging the tubes at 12,000×g for 5 min. The protein content was quantified using Genova nano (Jenway, UK) and about 75 μg of protein was subjected to SDS-PAGE (12–14%). Proteins in the slab gel were transferred to a PVDF membrane (Amersham, USA) which was blocked with 5% bovine serum albumin for western blot analysis. The membrane was then incubated overnight at 4 °C with HSP antibodies, namely, HSP 90 alpha and beta, HSP 70, HSP 60, and ubiquitin (Cell Signaling Technology, USA), and reacted with goat-anti rabbit conjugated with horseradish peroxidase. Diaminobenzidine was the substrate used for the development of color, and intensities of the protein bands were measured using ImageJ software.