Nbd s1p

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.

SphK activity was followed as phosphorylation of (7-nitro-2-1,3-benzoxadiazol-4-yl)-d-erythro (NBD)-sphingosine (Avanti Polar Lipids) to NBD-S1P as described previously39 (link) with modification using a UPLC (ultra performance liquid chromatography) system (Waters). Quantification was achieved by comparison with NBD-S1P (Avanti Polar Lipids) standards. Cell and tissue lysates were prepared as previously described7 (link). Values were expressed as percent of control.

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.