Animals were anesthetized using phenobarbital and perfused transcardially with phosphate-buffered saline (PBS). Brains were removed, with cortex, hippocampus, and striatum subdissected and then flash-frozen using liquid nitrogen until experiment. Brain samples were homogenized as described above, and protein concentration was measured by BCA assay. Tissue homogenate (500 μg of total protein) was mixed in immunoprecipitation (IP) buffer containing 50 mM tris (pH 7.4), 150 mM NaCl, 1 mM MgCl2, 1 mM EDTA, 1% NP-40, 0.25% SDS, and a protease inhibitor mix with either protein-specific antibody or whole immunoglobulin G (IgG) (both 1 μg) prebound to protein G magnetic beads (2 hours) at 4°C on a rotator for 2 hours. Protein G magnetic beads were then isolated by magnet and washed three times with IP buffer, followed by elution in SDS sample buffer, and boiled at 100°C for 10 min. Samples were analyzed by immunoblotting, as described above.

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