HEK293T cells were homogenized with homogenization buffer [5 mM Hepes (pH 7.4), 1 mM EDTA, 250 mM sucrose, and protease inhibitor cocktail], and supernatant was prepared by centrifugation at 800g. After centrifugation at 100,000g for 1 hour at 4°C, the pellet was solubilized with CHAPS buffer [2% CHAPS, 50 mM tris (pH 7.5), 2 mM EDTA, and 150 mM NaCl]. The soluble lysates were centrifuged again at 100,000g for 30 min, and the supernatants were subjected to glycerol gradient centrifugation. The same amount of protein extracts was applied to the top of 10 to 40% (w/v) linear glycerol gradient and centrifuged for 15 hours at 100,000g and 4°C using a Beckman SW 32.1 Ti rotor. Each fraction was collected from the top to the bottom of the gradient, and the same volume of each fraction was analyzed by Western blotting.

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