Cells were rinsed twice with PBS, pelleted and resuspended in 1 ml TEN buffer containing Complete protease inhibitor cocktail followed by homogenization. The cell lysates then were centrifuged at 3,000 rpm for 20 min at 4uC. Five hundred ml of the postnuclear supernatants were mixed with an equal amount of TEN buffer, and were then applied to the top of a pre-made 25-45% discontinuous sucrose gradient. This gradient was prepared in TEN buffer containing 1 ml of each of 25%, 35%, and 45% sucrose. The gradient was then centrifuged at 130,0006g for 1 hour at 4uC. Five 0.8-ml fractions were collected from the top of the centrifuge tubes. The proteins present in aliquots of each fraction were precipitated with 10% TCA and subjected to western blot analysis as described in the membrane flotation assay.