Tissue lysate preparation and Western blot analysis

Human post mortem brain (Table S3) or mouse brain tissue were homogenized and prepared in lysis buffer [50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton x-100, 0.5% SDS, 0.5% sodium-deoxycholate, phosphatase inhibitor mixture I and II (Sigma-Aldrich, St. Louis, MO), and complete protease inhibitor mixture (Roche, Indianapolis, IN)], using a Diax 900 homogenizer (Sigma-Aldrich). After homogenization, samples were rotated at 4°C for 30 min for complete lysis, the homogenate was centrifuged at 15,000 x g for 20 min and the supernatants were used for further analysis. Protein levels were quantified using the BCA assay (Pierce, Rockford, IL), samples were separated using SDS-polyacrylamide gels and transferred onto nitrocellulose membranes. The membranes were blocked with 5% non-fat milk in TBS-T (Tris-buffered saline with 0.1% Tween-20) for 1 h, probed using primary antibodies (Table S4) and incubated with appropriate HRP-conjugated secondary antibodies (Cell signaling, Danvers, MA). The bands were visualized by ECL substrate.