Brain Tissue Fractionation and Protein Extraction

Brain homogenates were prepared as previously described^{59 (link)}. Briefly, 200 mg of frontal cortex were homogenized in 5 volumes of 20 mM Tris-HCl, pH 7.5, 140 mM NaCl, added with Complete Protease Inhibitors cocktail and Phosphatase Inhibitors Cocktail 2 (Sigma) using a manual Dounce homogenizer and ultracentrifuged at 100,000 xg for 1 hour at 4 °C. The supernatant was collected, aliquoted and stored at −80 °C as the S1fraction. The pellet was re-homogenized in 1% Chaps, 1% Deoxycholate, 0.2% SDS, 140 mM NaCl, 10 mM Tris-HCl, pH 7.5, added with Protease and Phosphatase Inhibitors and ultracentrifuged at 30,000 xg for 30 minutes at 4 °C. The supernatant was aliquoted and stored at −80 °C as the S2 fraction; the pellet was homogenized in 2% SDS, 20 mM Tris-HCl, pH 7.5, 140 mM NaCl and ultracentrifuged at 30,000 xg for 30 minutes at 4 °C. The supernatant was saved as the S3 fraction and stored at −80 °C; the pellet was extracted in 4% SDS, 8 M Urea (P3 fraction). The total proteins amount was measured in each fraction by BCA Protein Assay kit (Pierce). Immunodepletion was carried out by using Protein G Mag Sepharose beeds (GE Healthcare) and a mixture of 4G8 and 6E10 antibodies.