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. 2005 Jul 18;202(2):249–259. doi: 10.1084/jem.20041685

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Copyright © 2005, The Rockefeller University Press

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Figure 7. — Ceramide generation in lipid rafts of WR/Fas-SM(−) and WR/Fas-SMS1 cells. (A) Ceramide contents in membrane fractions of sucrose density gradient fractionation. 10⁸ cells were lysed in Triton X-100 buffer and subjected to sucrose density gradient fractionation. Lipids of each fraction were extracted by the method of Bligh and Dyer (reference 53), and ceramide content was measured by the diacylglycerol kinase assay. After separation of ceramide-1-phosphates by TLC, radioactivity was visualized and estimated. The results are representative of three independent experiments and expressed as the percentage of total PSL arbitrary units. (B) Fas-mediated ceramide generation in lipid rafts. 10⁸ cells were left unstimulated or were stimulated with CH11 for 5 min and Triton X-100 lysates were subjected to sucrose density gradient fractionation. Lipids of raft fraction (fraction 4) were extracted by the method of Bligh and Dyer, and ceramide generation was measured by the diacylglycerol kinase assay. Radioactivity was visualized and estimated. The results are representative of three independent experiments. (C) The mean of three independent experiments revealed that ceramide contents of lipid rafts in WR/Fas-SMS1 cells were significantly greater than those of WR/Fas-SM(−) cells. The results were the mean of three independent experiments and expressed as the percentage increase of PSL arbitrary units. Error bars represent SEM. *, P < 0.01.