For measuring the level of active form of PPARα protein, a non-radioactive eletrophoretic mobility shift assay kit was used according to the manufacturer’s instructions (Panomics, Redwood, CA). Nuclear protein (4 μg) that was extracted from the brain tissue was incubated with poly d(I–C) in binding buffer for 5 minutes at room temperature, and then at 15°C for 30 minutes with biotinylated oligonucleotide containing the PPARα-binding site. The samples were separated by gel eletrophoresis using 6% polyacrylamide gel in 50 mM Tris buffer containing 45 mM boric acid and 0.5 mM EDTA at 4°C, blotted onto a Biodyne B (0.45μm) positively charged nylon membrane (Pall Corporation, Ann Arbor, MI), and then cross-linked for 3 minutes with an ultraviolet light. The biotinylated oligonucleotides were detected with streptavidin-horseradish peroxidase conjugate and the membranes were exposed to autoradiography film (Denville, Metuchen, NJ).
