FIGURE 4.

CERS2 enzymatic activity is highly dependent on phosphorylation. HEK 293T cells were transfected with a vector or a plasmid encoding HA-CERS1–6. Total membrane fractions (10 μg of protein) prepared from the transfected cells were treated with or without λPPase at 30 °C for 1 h, followed by incubation with 5 μm deuterium-labeled sphingosine and 25 μm of the indicated acyl-CoA at 37 °C for 30 min. Lipids were extracted, and deuterium-labeled ceramides were analyzed by a Xevo TQ-S LC/MS system and quantified by MassLynx software. The values represent the activity of each CERS relative to that of the vector/λPPase (−) sample and are the mean ± S.D. from three independent assays. Statistically significant differences are indicated; **, p < 0.01; t test.