Silica gel g 60

SK activity was determined under the conditions reported by Olivera and Spiegel (21) . SMC were homogenized in the previously used lysis buffer (23, 27) . The SK1 assay contained cell extract (100 mg protein), sphingosine (50 mM), [ 33 P]ATP and unlabeled ATP (1 mCi and 1 mM, respectively), 0.25% Triton X-100, 10 mM MgCl2, 20 mM Tris/HCl pH 7.4 (final volume 0.2 mL). After 30 min incubation at 378C, the reaction was stopped by 20 mL of 1 N HCl. [ 33 P]S1P was extracted, isolated by TLC on Silica Gel G60 (Whatman, Dominique Dutscher sas, Brumath, France) using butanol/ methanol/acetic acid/water (80/20/10/20), and the radioactive spots of [ 33 P]S1P were counted by liquid scintillation. Protein concentration was determined using the Bradford reagent (Biorad, Marnes La Coquette, France). Note that the SK assay containing detergents evaluates mainly SK1 activity (28) .