PSD protein purification.

We adapted a previous protocol for the preparation of postsynaptic density (PSD) fractions (13, 52). Forebrain cortical tissues were microdissected and Dounce-homogenized in 10 mL of homogenization buffer (0.32 M sucrose, 4 mM HEPES, pH 7.4 with protease inhibitors [Roche, cOmplete, EDTA-Free Protease Inhibitor Cocktail Tablets; 5056489001]). The homogenate was centrifuged at 1000 g for 10 minutes at 4°C to pellet cellular debris and nuclei (P1), and the subsequent supernatant (S1) was centrifuged for another 15 minutes at 10,000 g at 4°C. The resulting pellet (P2, “crude” synaptosomes) was resuspended in another 10 mL of homogenization buffer and centrifuged at 10,000 g for 15 minutes at 4°C. The supernatant was discarded, and the resulting pellet (P2′) was resuspended in 10 mL of 4 mM HEPES (pH 7.4), then homogenized on ice. The lysate was incubated at 4°C for 30 minutes while shaking to hypoosmotically lyse the synaptosomes and then centrifuged for 20 minutes at 25,000 g at 4°C. The resultant pellet (LP1) was resuspended in 1 mL of homogenization buffer and layered on top of a discontinuous sucrose gradient (bottom to top: 1.5 mL of 1.2M sucrose, 1 mL of 1.0M sucrose, and 1 mL of 0.8M sucrose). The gradient was ultracentrifuged at 150,000 g for 1.5 hours at 4 °C. The turbid layer between the 1.0M/1.2M sucrose interphase containing the synaptic plasma membranes (~1 mL) was collected and resuspended in 5 mL of 4 mM HEPES to dilute out the sucrose. This fraction was ultracentrifuged again at 200,000 g for 30 minutes at 4°C. The resulting pellet was resuspended in 1 mL of 50 mM HEPES with 2 mM EDTA (pH 7.4) and the membrane proteins extracted by adding Triton X-100 at a final concentration of 0.5% and incubating at 4°C while rotating for 15 minutes. The proteins were centrifuged at 32,000 g for 20 minutes at 4°C and the resulting pellet resuspended in 75 μL of 50 mM HEPES with 2 mM EDTA.