Phosphorylated tauser396

The hippocampus samples were lysed in 20 mM Tris buffer (pH 7.4) containing 2 mM EDTA, 137 mM NaCl, 1% NP40, 10% glycerol, and 12 mM α-glycerol phosphate and protease inhibitors. After 30 min on ice, the lysates were centrifuged for 10 min at 11,300× g at 4 °C. After measuring the protein contents in the lysates using a Bio-Rad protein assay kit, lysates with equal amounts of protein were immunoprecipitated with specific antibodies before separation by SDS-PAGE as previously described [13 (link)]. The antibodies used for immunoblot analysis were cAMP responding element-binding protein (CREB), phosphorylated CREB^ser133, Akt, phosphorylated Akt^Ser473, GSK-3β, phosphorylated GSK-3β^ser9, tau, phosphorylated tau^ser396, and β-actin (Cell Signaling Technology). The intensity of protein expression was determined using Imagequant TL (Amersham Biosciences, Little Chalfont, England).

Hippocampal tissues from four rats from each group were dissected as previously described.^{(22 (link))} Each tissue was lysed with RIPA lysis buffer with added protease inhibitors, and their protein contents were measured using a Bio-Rad protein assay kit (Hercules, CA). The lysates with equivalent amounts of protein (30–50 µg) were resolved into sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the amounts of proteins of interest were examined by immunoblotting with the specific antibodies as follows: protein kinase B (PKB or Akt), phosphorylated PKB^Ser473, forkhead box protein O1 (FOXO1), phosphorylated FOXO1^Ser256, phosphorylated tau^Ser396, tau (Cell Signaling Technology, Danvers, MA) and β-actin (Santa Cruz Biotech, Dallas, TX). The intensity of the proteins detected were measured using Imagequant TL (Amersham Biosciences, Piscataway, NJ).