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Fig. 3. Subcellular fractions of human fibroblasts derived from an FHC patient (A), HEK (B) cells or CV1 cells (C) overexpressing full-length human MK were obtained by Nycodenz equilibrium density gradient centrifugation as described in Materials and Methods. Fractions were analysed for the cytosolic marker PGI (black bars), the peroxisomal marker CAT (grey bars) and MK (open bars). Relative activities were expressed as a percentage of total gradient activity present in each fraction. The patterns of distribution of MK activity and MK protein as determined by immunoblot analysis with an affinity purified antibody raised against human MK were similar to the pattern of PGI activity. Human fibroblasts derived from an FHC patient (D), HEK cells (E) or CV1 cells (F) overexpressing full-length human MK were incubated with increasing concentrations of digitonin as described in Materials and Methods. Supernatant (open symbols) and pellet (closed symbols) fractions were analysed for the activities of the cytosolic marker PGI (square), the peroxisomal marker CAT (triangle) and MK (circle). Relative activities were expressed as a percentage of total activity (supernatant + pellet) present in each fraction. The patterns of latency of MK activity and MK protein as determined by immunoblot analysis with an affinity purified antibody raised against human MK were similar to the pattern of PGI activity.
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