Preparation of striatal lysates for sucrose gradient centrifugation experiments

Adult mice were killed by decapitation and brains rapidly removed. Striatum from PICK1 wild-type and KO mice were rapidly dissected and homogenized in homogenization buffer (0.32 M sucrose and 4 mM HEPES; pH 7.4). Samples were centrifuged at 1000 × g for 10 min at 4°C to remove cell nuclei followed by centrifugation at 16,000 × g for 20 min at 4°C to pellet the membranes. The pellet was homogenized in 1-ml lysis buffer [1% Brij56 (w/V)], protease inhibitor, and 0.2 mM PMSF in the gradient buffer (25 mM HEPES and 150 mM NaCl; pH 7.4) for 15 min on ice. A 1-ml sample was mixed with 1-ml sucrose (80% w/V) in gradient buffer to create 2 ml with final 40% (w/V) sucrose content in a 15-ml centrifuge tube (Beckman Coulter). On top of this, a 12 ml 35–15% continuous gradient was assembled using a SG15 gradient maker (Hoefer). Subsequently the gradient was ultracentrifuged at 100,000 × g for 18 h in a Beckman SW28.1 rotor head (Beckman Coulter) and fractionated into nine fractions using a P1 pump. Each fraction was incubated for 30 min at 37°C in SDS loading buffer with a final concentration of 66 mM DTT followed by immunoblotting.