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. 2012 Aug;53(8):1701–1707. doi: 10.1194/jlr.D025627

Fig. 1.

Fig. 1.

Synthesis of C16:0 or C24:1 NBD-dihydroceramide catalyzed by endogenous or overexpressed ceramide synthases. A: TLC image illustrating that CerS reaction proceeds only in the presence of cell lysate and fatty acid-CoA. Homogenates of untransfected (lanes 1, 2, 4, and 8), CerS2- (lanes 5 and 9), or CerS5-expressing (lanes 6 and 10) HEK293 cells (25 μg/reaction) were incubated for 30 min with 10 μM NBD-sphinganine and 50 μM C16:0-CoA (lanes 3–6) or C24:1-CoA (lanes 7–10). Lane 1: no NBD-sphinganine control; Lane 2: No fatty acid-CoA control; Lanes 3 and 7: no homogenate control (i.e., no enzyme). B: Product formed as a function of lysate concentration, using (i) C16:0-CoA or (ii) C24:1-CoA as the fatty acid substrate. Reactions in B(i) were run for 30 min while those in (ii) were run for 60 min. C: Product formation as a function of time, using CerS5-expressing cell homogenate (5 μg/reaction) and C16:0-CoA (50 μM), or CerS2-expressing homogenate (50 μg/reaction) and C24:1-CoA (50 μM). Data points are mean and standard error of triplicate assays and representative of two or more independent experiments.