Fig. 5.

Comparison of CerS assays using TLC or SPE columns. A: Homogenates of nontransfected HEK cells were assayed in 20 μl reaction volume with increasing amounts of protein with 15 μM NBD-Sph, 50 μM C16-CoA (upper) or C24:1-CoA (lower), and 20 μM defatted BSA, at 37°C. NBD-Sph and NBD-ceramide were separated by TLC (gray) or by SPE columns (black). B: Homogenates of CerS5-overexpressing cells were assayed as in A with C16-CoA. Results are ± SD of a typical experiment repeated three times with similar results. C: Homogenates (10 μg of protein in 20 µl) were prepared from CerS5-overexpressing cells and were assayed for increasing amounts of time. Results are means ± SEM of two to three independent experiments.