Spinal cord tissue was isolated as for WB, and the SDH of spinal L3 to L5 were isolated. The procedure for fractionation of spinal cords from mice into synaptosomes, synaptic vesicle fractions, synaptic plasma membrane, and postsynaptic densities was performed, as previously described (6). All procedures were performed at 4°C. Briefly, spinal cords were isolated from eight WT and eight Sort1−/− mice (8 to 12 weeks of age), pooled, and homogenized following addition of 10 volumes of ice-cold 0.32 M sucrose, 4 mM Hepes, and protease inhibitor (pH 7.4; cOmplete, Roche), using a glass Teflon homogenizer. WT and Sort1−/− samples were processed in parallel for direct fraction comparison. After centrifugation of the homogenate for 10 min at 1000g, P1 and S1 corresponded to pellet and supernatant, respectively. S1 was further centrifuged for 15 min at 10,000g to obtain supernatant S2 and pellet P2, a crude synaptosomal preparation. Solubilized P2 was centrifuged for 15 min at 10,000g, and the resulting pellet was lysed by hypoosmotic shock in H2O, rapidly adjusted to 4 mM Hepes (pH 7.4), and stirred for 30 min. The lysate was centrifuged at 25,000g for 20 min, generating the synaptosomal membrane fraction P3 and a supernatant S3 enriched in presynaptic vesicles. S3 was further centrifuged at 165,000g for 2 hours, generating pelleted synaptic vesicle preparation enriched in synaptic vesicles. Solubilized P3 was applied to a discontinuous sucrose gradient containing 0.8, 1.0, and 1.2 M sucrose and centrifuged at 150,000g for 2 hours. The fraction between the 1.0 and 1.2 M sucrose layers was recovered and diluted to 0.32 M sucrose, after which it was centrifuged again at 150,000g for 30 min, resulting in the pelleted synaptosomal plasma membrane (SPM) fraction. The SPM fraction was resuspended with 0.4% Triton X-100 in 50 mM Hepes (pH 7.4) and 2 mM EDTA containing protease inhibitors and was centrifuged at 35,000g for 20 min to obtain the pelleted postsynaptic density fraction I (PSDI) containing postsynaptic densities. Purity was increased by resolubilizing the PSDI fraction, followed by centrifugation at 200,000g for 20 min yielding PSDII. The total protein concentration in the fractions was determined using a bicinchoninic acid kit (Sigma-Aldrich). Equal amounts of proteins of each fraction were separated by reducing SDS-PAGE and analyzed by WB for the presence of TrkB (1:1000; AF1494, R&D Systems), PSD95 (1:1000; P-246, Sigma-Aldrich), and synaptophysin (1:1000; MAB5258, Chemicon).

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