Preparation of HMW extract

Cultured cell pellets resuspended in ten volumes of cold hypotonic buffer (10 mM HEPES-KOH pH 7.5, 1.5 mM MgCl₂, 10 mM NaCl, 1.25× cOmplete^TM protease inhibitors (Roche), 0.5 mM dithiothreitol (DTT), 0.15% NP-40) or mouse tissues homogenized in cold brain lysis buffer (10 mM HEPES-KOH pH 7.5, 5 mM CaCl₂, 3 mM MgCl₂, 0.1% NP-40, 0.1 mM EDTA, 0.32 M Sucrose, 1 mM DTT) by pushing through 26 G needles were centrifuged at 956 × g for 8 min at 4 °C followed by incubation with ten volumes of HMW lysis buffer (20 mM HEPES-KOH pH 7.5, 150 mM NaCl, 1.5 mM MgCl₂, 0.5 mM DTT, 1.25× cOmplete^TM protease inhibitors, 0.6% Triton X-100) for 5 min. Soluble fractions and pellets were separated by centrifugation at 20,000 × g for 5 min at 4 °C. The pellets were lysed with an equal volume of HMW lysis buffer containing 5 U/µl of Benzonase (Sigma, E1014-25KU) at 25 °C on a rotator for 20 min, and the HMW extracts were cleared by centrifugation at 20,000 × g for 10 min at 4 °C.