Fig. 2. Detection of nSMase 1 in subcellular fractions of AH7974 cells. (A) Scheme for isolating subcellular fractions of AH7974 cells (for details, see Materials and Methods). Pellets obtained by centrifugation’s at 8000 g and 105,000 g were designated as mitochondrial and microsomal fractions, respectively. They may contain some of other organelle but were not purified further. Nucleus and plasma membrane were highly purified by this protocol, as shown in the electron microscopic photographs (B). (C) Subcellular fractions of AH7974 (50 µg protein each) were subjected to western blotting with the anti-rnSMase 1 antibody. An immunoreactive protein band was detected at 47.6 kDa. The microsomal marker Grp78 was also detected with an anti-KDEL antibody in the same way. Extracts of whole cells and organelle showed the bulk activities of neutral SMases (nmol/mg protein/hour) as follows: whole cell, 17.7; nucleus, 5.80; mitochondria, 20.4; microsome, 26.3; plasma membrane, 22.2.