Sph kinase 1 and Sph kinase 2 assays

NBD-C₁₈-shingosine (Avanti Polar Lipids) was prepared and dissolved in 5% Triton X-100, and Sph kinase (SphK1) and SphK2 assays were performed in real-time as previously described (33) with slight changes. Briefly, 96-well polypropylene plates were used. Fluorescence emission was measured with a FLUOstar Omega (BMG Labtech). Excitation wavelength was 544 nm, and emission wavelength was 590 nm. Assays were initiated with 20× ATP [20 mM ATP, 200 mM MgCl₂, 900 mM Tris-HCl (pH 7.4)] followed by shaking and mixing for 15 s. Assays were prepared as master mixes immediately before use in either SphK1 or SphK2 reaction buffer containing 30 μM of NBD-Sph, 150 nM of rhSphK1 (R&D Systems, 5536-SK-010), or 6.9 nM of rhSphK2 (R&D Systems, 5298-SK-010). ARC39 was added as indicated. SphK1 reaction buffer contained 30 mM of Tris-HCl (pH 7.4), 0.05% Triton X-100, 150 mM of NaCl, 10% glycerol, 1 mM of Na₃VO₄, 10 mM of NaF, and 10 mM of β-glycero-phosphate. SphK2 reaction buffer contained 30 mM of Tris-HCl (pH 7.4), 0.05% Triton X-100, 200 mM of KCl, and 10% glycerol.