Sucrose gradient fractionation was performed as described previously (63). Cells were suspended in buffer S [25 mM tris (pH 7.4), 150 mM NaCl, 2 mM EDTA, 1% CHAPS, and protease inhibitor cocktail] and homogenized by 10 passages through a 25-gauge needle. After centrifugation at 10,000g for 30 min, soluble lysates were adjusted to a final concentration of sucrose (45%) and transferred to a 10-ml ultracentrifuge tube. Then, a discontinuous sucrose gradient was formed by sequentially layering 35% sucrose (3.2 ml) and 5% sucrose (3.2 ml), and the tubes were subjected to ultracentrifugation at 100,000g for 16 hours in Beckman SW 32.1 Ti rotor at 4°C with no brakes. Twelve 0.8-ml fractions were 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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