Proteins from brain homogenate or cortical nuclear extracts were prepared as previously described (48, 59). Proteins were resolved in 7.5-10% SDS-polyacrylamide gels, and immunoblotted with antibodies to phospho-Ser256-FoxO1, FoxO1, phospho-Ser253-FoxO3a, phospho-Thr308-Akt, phospho-Ser473-Akt, Akt (Cell Signaling Technologies, Danvers, MA), phospho-Thr32-FoxO3a, FoxO3a, CREB (Upstate Biotech, Lake Placid, NY), and β-tubulin (Sigma-Aldrich, St. Louis, MS). Following a reaction with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG, the immunoreactions were detected by enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ), and protein bands were quantified with a densitometer.
