Sphingosine kinased-erythro-sphingosineSphingosine-1-phosphateThin-layer chromatographyBligh–Dyer extractionSphingosine kinases (SK) 1 and 2 are unique lipid kinases that phosphorylate sphingosine to form -sphingosine-1-phosphate (S1P). S1P is a bioactive molecule eliciting multiple effects both ...
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Shanghai, China) assay was used to determine cell viability. For CCK8 assay, 24 h after OGD, 10 μl CCK8 was added into each well of 96-well plates, which were incubated at 37 °C for 2 h. Optical density was measured at 450 nm using a plate reader (ELX 800; Bio-...
Sphingosine kinase activity assay SK activity was measured as described by Olivera and colleagues [17] with a few modifications, as described previously [10]. Specific activity of SK was expressed as picomoles of S1P formed per minute per milligram of protein. Cell transfection C2C12 cells were ...
Measurement of Sphk1 activity 0.1 g tissue extracts from liver were used for Sphk1 activity assay with a commercial kit according to the manufacturer’s instructions (Sphingosine Kinase Activity Assay Kit, Echelon Biosciences Inc.). Statistical analysis Data are presented as mean ± SEM. Compar...
To each 50 μL kinase activity assay, 10 μM C17 Sph was added as sub- strate. Reactions were started with the addition of 5 μg of protein lysate, and the reaction mix was incubated at 37°C overnight. Reactions were stopped with 200 μL of metha- nol containing 100 pmoles d18:0...
A, agarose-bound cardiolipin or control phosphatidylethanolamine (PE) and phosphatidylcholine (PC) beads were co-incubated with 1 nmol of SPH at the indicated pH, extensively washed, and extracted, and the amount of bound sphingosine was determined by a sphingosine kinase assay. The solvent octyl...
Billich A, Bornancin F, Mechtcheriakova D, Natt F, Huesken D, Baumruker T: Basal and induced sphingosine kinase 1 activity in A549 carcinoma cells: function in cell survival and IL-1beta and TNF-alpha induced production of inflammatory mediators. Cell Signal. 2005, 17: 1203-1217. 10.10...
(125 mM Tris, pH 6.7, 0.5 mM Na4P2O7, 1.25 mMEDTA, 0.5% w/v SDS containing 1.25% v/v glycerol, 0.06% w/v bromophenol blue and 50mM dithiothreitol) and were subjected to SDS-PAGE and Western blotting as previouslydescribed (12).Sphingosine Kinase Activity AssaySK2 activity was ...
In vivo SPHK activity assay Sphingosine kinase activity was measured as described previously56. Briefly, the labeled S1P was separated by TLC on Silica Gel G-60 (GE Healthcare, Little Chalfont, UK) with 1-butanol/ethanol/acetic acid/water (80:20:10:20, v/v) and visualized by autoradiograph...