Total lysate and synaptoneurosome (SN) were prepared from the hippocampus. The isolated hippocampus was homogenized with 0.32 M sucrose in 4 mM HEPES solution. The lysates were then centrifuged at 1000 × g for 10 min at 4 °C. The supernatant was transferred to the new collection tube and centrifuged at 10,000 × g for 15 min at 4 °C. The resulting supernatant (total lysate) was stored, and the remaining pellets were re-suspended with 0.32 M HEPES buffered sucrose solution. The re-suspended sample was centrifuged at 10,000 × g for 15 min at 4 °C. To lyse the sample, the pellets were re-suspended in dd H2O in order to provide a hypo-osmotic shock, transferred to a glass–Teflon tissue homogenizer, and homogenized rapidly by hand (3 strokes). The sample was transferred to a new collection tube, suspended in 4 mM HEPES solution prepared by the addition of 1 M HEPES, and rotated for 30 min at 4 °C to ensure complete lysis. The lysed sample was centrifuged at 25,000 × g for 20 min at 4 °C, and then the supernatant was removed. Pellets were re-suspended in 0.32 M HEPES buffered sucrose solution (synaptoneurosome fraction) and subjected to western blotting.

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