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2, 0.005% Triton X-100, 10 mM KCl, 10 mM NaF, and 1.5 mM semicarbazide. Following incubations for different time points up to 2 h, lipids were extracted and separated by thin-layer chromotography (TLC) plates (Merck) using butanol/acetic acid/water 3:1:1 as mobile phase Radiolabeled derivatives were visualized and quantified using a Molecular Dynamics Storm PhosphorImager (Sunnyvale, CA). The calculated phosphorylation efficiency is reported as value relative to sphingosine (=100%) for which the rate was 41 and 25 nmol/min/mg with SPHK1 and SPHK2, respectively.
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50 were performed using a nonlinear regression fit program provided in the Origin 7 RS2 software package (Origin LabCorporation).
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