FIGURE 3.
In vitro SMS activity and in vivo C6-NBD-ceramide utilization. A, homogenates of WT and ldlC cells were assayed for the activities of SMS and glucosylceramide synthase in conditions of linearity with time and protein concentration. Lipids were extracted from incubates, separated by HPTLC, and formed fluorescent compounds visualized under UV light illumination. Quantification was carried out by densitometry of the fluorescent bands using ImageJ software. Enzyme activity values are given as mean arbitrary fluorescent units ± S.D. for three independent experiments. Other details were as described under “Experimental Procedures.” B, C6-NBD-ceramide (Cer) utilization in ldlC cells. WT and ldlC cells monolayers were cultured in the presence of 1 μg/ml C6-NBD-ceramide for 4 h. Cells were harvested, and lipids were extracted and separated by HPTLC. Formed fluorescent compounds were photographed under UV light illumination, and the percent contribution of the bands quantified by densitometry using ImageJ software. For A and B, the positions of lipid standards are indicated at the left of the chromatogram.