Sphingosine Kinase 1 Enzymatic Assay

We confirmed S1P formation through SphK1 enzymatic activity measurements as previously described^{11 (link)} using a UPLC (Ultra performance liquid chromatography) system (Waters). Briefly, 3 μl of SphK1 (Cayman, 10348) were mixed with 3 μl of assay buffer (200 μM NBD-sphingosine (Avanti Polar Lipids), 100 mM of HEPES buffer, pH 7.2, 10 mM MgCl₂, 200 mM Semicarbazide, 1 mM 4-deoxpyridoxin, 2 mM Dithiothreitol, and 0.2% Igepal CA-630, all from Sigma-Aldrich) in the presence of 0–50 mM acetyl-CoA (Sigma-Aldrich, A2056), and the mixture were pre-incubated at 37 °C for 10 min. After pre-incubation, 1 mM ATP (Amresco, 0220) was added the mixture, and it was incubated at 37 °C for 1 h. The hydrolysis reactions were stopped by adding 53 μl of ethanol, and centrifuged at 15,493×g for 5 min. Thirty microliter of the supernatant was then transferred to a sampling glass vial and 5 μl was applied onto a UPLC system for analysis. S1P formation was followed as phosphorylation of (7-nitro-2-1,3-benzoxadiazol-4-yl)-d-erythro (NBD)-sphingosine (Avanti Polar Lipids) to NBD-S1P. Quantification was achieved by comparison with NBD-S1P (Avanti Polar Lipids) standards.