Fig. 4. Identification of nuclear neutral SMase as nSMase 1. (A) Immuno-precipitation of the neutral SMase activity in extracts from nuclei and plasma membranes with anti-rnSMase 1 antibody. Extracts were prepared as described in Materials and Methods. Aliquots (20 µl) containing activity of neutral SMase (2.5 nmol/hour) were mixed with the anti-rnSMase 1 antibody, and antigen-antibody complexes were adsorbed onto protein A-Sepharose beads. After the incubation, the mixture was centrifuged and supernatants were assayed for neutral SMase activity, indicated as% of the control. B. Nuclear extract (10 ml) from AH7974 cells was applied to a Sephacryl S-300 column (1.2 x 85 cm) and 7 ml fractions were collected. Activity of neutral SMase was assayed with 10 µl aliquots of each fraction, and the peak fractions (10 µl each) were subjected to Western blotting using anti-rnSMase 1 antibody. As a molecular size marker, histidine-tagged nSMase 1 (49.4 kDa) was shown in the first lane. Open circles, optical density at 280 nm; closed circles, neutral SMase activity.