Grinding kit

Tissue homogenization and 1-DE protein separation were done as previously described (11) .
Briefly, 100 mg of each tissue was homogenized in a detergent solution (4% Triton X100, 1X antiprotease cocktail; Sigma, St Louis, MO, USA) and ground using a grinding kit (GE Healthcare) before protein precipitation with a 2D cleanup kit (GE Healthcare). The supernatant was removed and the pellet was resuspended in 250 ml of sample buffer (8 M urea/2 M thiourea [Sigma], 4% CHAPS [Sigma]). Protein concentration was determined using the Bradford assay (BioRad). Proteins (500 mg per gel) were eluted into rehydration buffer (8 M urea/2 M thiourea [Sigma], 2% CHAPS [Sigma],
DeStreak reagent [15 mg/ml, GE Healthcare] and ampholytes [1% IPG buffer, GE Healthcare]) before first separation according to their isoelectric points along a nonlinear immobilized pH-gradient (IPG) strip (pH 3-11 NL, 18 cm long) using an IPGphor III apparatus (GE Healthcare), as described elsewhere (14) . For the second dimension, equilibrated strips were loaded onto 8-18% SDSpolyacrylamide gels and electrophoresis was performed as in (19) . One preparative gel was stained with CBB G-250 (Sigma) and used for spot cutting and protein sequencing. The remaining gels were electroblotted onto ECL membranes (GE Healthcare).

Brain regions were homogenized using pestles from the Grinding Kit (GE Healthcare, Chicago, Illinois, USA) in core buffer as previously described 20 supplemented with 1 Complete Mini Protease Inhibitor Cocktail tablet (Roche, Penzberg, Germany) per 10 ml buffer. The homogenate was split in two fractions for total cell lysate and for enrichment of synaptosomes, respectively. The total cell lysate was prepared by two repeated freeze-thaw cycles, bath sonication and centrifugation at 12000 rpm for 5 min. The supernatant was isolated and stored at -20°C upon further use. The synaptosomal fraction was prepared as described before with minor adaptations 20 . In short, the homogenate was filtered through a 5 µm membrane (Millipore, Billerica, Massachusetts, USA). Thereafter the flow-through was centrifuged to pellet the synaptosomal fraction. The frontal cortex samples for MS analysis were redissolved in core buffer. The frontal cortex (for validation), hippocampal and striatal samples were redissolved in 0.1% SDS in core buffer and sonicated at maximum intensity for 1 min. Total protein concentration of all