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Fig. 5. The subcellular localization of Bro1 requires ESCRT-III components. (A,B) Subcellular fractionation and western blotting was performed as described in the legend to Fig. 4A. (C) The P13 fraction was collected from vps4{Delta} cells, then resuspended in buffer containing 1% Triton X-100 and separated by centrifugation at 100,000 g for 1 hour into a soluble fraction (S100) and a pellet fraction (P100). The total protein content of each fraction was resolved by SDS-PAGE and examined by western blotting.