Preparation of subcellular fractions from rat brain and western immunoblotting

Brains from adult rats (7–12 weeks of mixed gender) were supplied by Rockland Immunochemicals Inc. (Gilbertsville, PA). Animals were anesthetized with isoflurane, brains were collected within two minutes after cervical dislocation and were immediately frozen in liquid nitrogen. Brains were rapidly thawed in isotonic sucrose solution and dissected to remove white matter. Cerebral cortices were homogenized in isotonic sucrose. Subcellular fractionation was essentially as described previously [14]. Homogenates were centrifuged at 900 g for 10 min and pellets (P1) containing nuclei were discarded. The supernatants (S1) were centrifuged at 10,500 g for 12 min to obtain P2 and S2 fractions. S2 fraction was further centrifuged 82,700 g for 2 h to obtain P3 (light membranes) and S3 (cytosolic) fractions. P2 was fractionated on a sucrose gradient to isolate synaptosomes. Synaptosomes were treated with 0.5% TritonX-100 and the detergent insoluble pellet was fractionated on a sucrose gradient. Crude PSD fraction from the 1.5/2.1M sucrose interface was extracted with 0.5% TritonX-100, 75 mM KCl and collected on a sucrose cushion. Proteins from subcellular fractions were resolved by SDS-PAGE using 4–15% Mini-PROTEAN TGX Precast polyacrylamide gels (BioRad). Gels were transferred to PVDF membranes using the Trans-Blot Turbo Transfer System (BioRad), blocked, incubated with primary and secondary antibodies, and visualized via chemiluminescence.