FIGURE 2.

PGN induced nSMase, but not aSMase, activation in RAW 264.7 macrophages. A, cells were incubated with PGN (30 μg/ml) for the indicated time intervals. Whole cell lysates were prepared. The activities of nSMase and aSMase were measured as described under “Experimental Procedures.” The results are representative of three independent experiments. B, cells were pretreated with 3-OMS (30 μm) and imipramine (30 μm) for 30 min and incubated with PGN (30 μg/ml) for another 20 min. After incubation, whole cell lysates were prepared. The nSMase activity was measured as described under “Experimental Procedures.” The results are expressed as the means ± S.E. of three independent experiments performed in duplicate. *, p < 0.05, compared with the PGN alone group. C, cells were transfected with 0.5 μg of RasN17 for 24 h or pretreated with 10 μm 3-OMS for 30 min and then incubated with 30 μg/ml PGN for another 24 h. Whole cell lysates were prepared and immunodetected with COX-2 or an α-tubulin-specific antibody. The presence of equal amounts of protein loading was confirmed by α-tubulin. The results shown are representative of three independent experiments. The results are expressed as the means ± S.E. *, p < 0.05, compared with the PGN-treated group.