Mouse brain regions of interest (i.e., striatum, cortex, and hippocampus) were homogenized in homogenization buffer containing 10 mM tris (pH 7.5), 500 mM NaCl, 10% sucrose, 1 mM EDTA, and a protease inhibitor mix (03969-34, Nacalai Tesque Inc.), followed by brief sonication. Tissue homogenates were centrifuged at 1500g for 5 min at 4°C to remove debris, with protein concentration measured using bicinchoninic acid (BCA) reagent (23227, Thermo Fisher Scientific Inc.) using bovine serum albumin (BSA) as standard. Ten micrograms of total proteins per lane was separated by SDS-PAGE in precast 5 to 20% gradient acrylamide gels (2331730, Atto Corp.) and transferred to polyvinylidene difluoride (PVDF) membrane by semidry electrophoretic transfer. PVDF membranes were blocked using 1:1 mix of tris-buffered saline with 0.05% Tween 20 (TBS-T) and Blocking One (03953-95, Nacalai Tesque Inc.) and then incubated with primary antibodies diluted using TBS-T/Blocking One overnight with gentle agitation at 4°C overnight. PVDF membranes were washed three times with TBS-T and incubated with secondary antibodies using TBS-T/Blocking One for 2 hours with gentle agitation at 4°C. After incubation with secondary antibodies, PVDF membranes were washed three times with TBS-T. Antibodies were detected using luminol-based chemiluminescence assay kits [Chemi-Lumi One L (07880) or Chemi-Lumi One Ultra (11644), Nacalai Tesque Inc.] and ImageQuant LAS 3000 Mini (GE Healthcare). No manipulations other than global contrast and brightness adjustments were performed on gel images in Adobe Photoshop. Primary antibodies used in this study are listed in table S2.

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