4.5. Enzyme Activity Assay for Ceramide Synthases

The ceramide synthases (CerSs) activity assay was performed as described previously [13,24]. Briefly, cell lysates, prepared in an assay buffer (20 mM HEPES, pH 7.4, 25 mM KCl, 2 mM MgCl2, 0.5 mM DTT, 0.1% (w/v) fatty acid-free BSA, and 50 μM fatty acid-CoA (C16:0, C18:0, C20:0, C22:0, C24:0, and C26:0)) were incubated with 10 nmol of C17-sphinganine (Avanti Polar Lipids, Alabaster, AL, USA) for 30 min at 37 °C. Total lipids were extracted by the addition of CHCl₃:MeOH (1:2, v/v), and 100 pmol of d17:1/C18:0 ceramide (Avanti Polar Lipids, Alabaster, AL, USA) was used as the internal standard, and applied onto the LC-ESI-MS/MS system (API 3200 QTRAP mass, AB/SCIEX, Framingham, MA, USA), as described previously [24]. The activity of CerS is expressed as pmol (d17:0 dihydroceramide production) per mg protein per min.