Brains were homogenized on ice using a glass potter in a 9-fold volume of homogenization buffer: 25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA containing phosphatase inhibitors (30 mM NaF, 0.2 mM Na3VO4, 1 nM Okadaic acid, 1 mM PMSF, 5 mM Na4P2O7) and a cocktail of protease inhibitors (Roche). Protein concentration was measured using Micro BCA Protein assay kit (Thermo Fisher Scientific). Protein samples were aliquoted and stored at −80 °C.