Brain RNA was extracted with Trizol Reagent (Invitrogen Life Technologies, Carlsbad, CA) and chloroform, precipitated with isopropanol, washed with 75% ethanol, and stored at -80°C in DEPC-treated water. RT-PCR was performed using the SuperScript III One-step RT-PCR system (Invitrogen Life Technologies, Carlsbad, CA) with the forward and reverse primers of FoxO1 (5′-CCTGTCGTACGCCGACCTCATCAC-3′ and 5′-GTCCATGGACGCAGCTCTTCTCCG-3′). PCR products (15 μl) were separated on a 2% agarose gel, visualized by ethidium bromide staining, and photographed with Fluor-S MultiImager (Bio-Rad, Hercules, CA).
