Three to four month old mice were deeply anesthetized with Isoflurane and sacrificed by decapitation. The hippocampi were collected on an ice-cold preparation table in 0.9% saline solution and stored at −80°C. All samples were processed in parallel. The samples were homogenized in ice-cold RIPA buffer (50 mM Tris-HCl, 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mM Na3VO4, 1 mM NaF) with 1 mg/mL protease inhibitor cocktail (aprotinin, leupeptin, pepstatin) (Roche, Cat # 11836153001). The homogenates were centrifuged at 14,000rpm for 30 min at 4°C and supernatant was collected. The protein concentration in the supernatant was determined using BCA Protein Assay kit (Pierce, Rockford, IL). Equal amounts of protein were treated with 2X Laemmli Sample Buffer (Bio-Rad, CAT#161-0737, CA) and 1X PBS without Calcium or Magnesium, pH 7.4 (Gibco-Life Technologies, CAT#10010023), incubated at 95°C for 5 min, and 16 μL (1.25 mg/mL) of total protein per lane was loaded onto 12.5% Tris-Glycine gel. Gel electrophoresis was performed at 100 V for 2 h with 10% Tris-Glycine running buffer (100 μL 10X Tris-Glycine, 10
