Fig. 1. Specific detection of nSMase 1 by western blotting using a rabbit polyclonal antibody. (A) Detection of GST-tagged nSMase 1 expressed in E. coli with immune rabbit serum. Lysate of E. coli expressing GST-tagged recombinant nSMase 1 was electrophoresed on 8% SDS-polyacrylamide gel, transferred to a membrane, and probed with immune rabbit serum (dilution 1:1000, lane 2), as described in Materials and Methods. An immunoreactive protein band was detected at 76.1 kDa, the predicted value for the GST-tagged nSMase 1. As a control, preimmune rabbit serum was used (lane 1). (B) Western blot analysis of the whole cell lysates and a nuclear extract using an affinity-purified anti-rnSMase 1 antibody. Whole cell lysates of AH7974 (lane 3) and 3Y1 (lane 5) were analyzed by western blotting using an affinity-purified antibody. Histidine-tagged nSMase 1 (49.4 kDa, lane 1) and GST-conjugated nSMase 1 (76.1 kDa, lane 2) were also stained as positive controls. Nuclear extract of AH7974 was also analyzed (lane 4). The arrow indicates the immunoreactive band detected at 47.6 kDa, corresponding to the predicted value for the full-length rat nSMase 1.
