Quantification of Sphingosine Kinase Activity

The brains or cells were lysed in homogenization buffer containing 50 mM HEPES (GIBCO), 150 mM NaCl (Sigma-Aldrich), 0.2% Igepal (Sigma-Aldrich) and protease inhibitor (Calbiochem)^{26 (link)}. We performed the enzymatic activity measurements as previously described^{26 (link)} using a UPLC (Ultra performance liquid chromatography) system (Waters). Briefly, 3 μl of the samples were mixed with 3 μl of SphK (200 μM NBD-sphingosine (Molecular Probes), 100 mM of HEPES buffer, pH 7.2, 10 mM MgCl₂, 200 mM Semicarbodize, 1 mM 4-deoxpyridoxin, 2 mM Dithiothreitol, 1 mM ATP and 0.2% Igepal CA-630) assay buffer and incubated at 37 °C for 1 h. The hydrolysis reactions were stopped by adding 54 μl of ethanol, and centrifuged at 13,000 rpm for 5 min. Thirty microliters of the supernatant was then transferred to a sampling glass vial and 5 μl was applied onto a UPLC system for analysis. SphK activity was followed as phosphorylation of (7-nitro-2–1,3-benzoxadiazol-4-yl)-derythro (NBD)-sphingosine (Avanti Polar Lipids) to NBD-S1P. Quantification was achieved by comparison with NBD-S1P (Avanti Polar Lipids) standards.