Inhibition of mouse SphK2 expressed in baculovirus infected Sf9 insect cells assessed as remaining activity using sphingosine and [gamma-(32)P]-ATP at 100 uM by scintillation proximity counting
Inhibition of mouse SphK2 expressed in baculovirus infected Sf9 insect cells assessed as remaining activity using sphingosine and [gamma-(32)P]-ATP at 10 uM by scintillation proximity counting
Inhibition of mouse SphK2 expressed in baculovirus infected Sf9 insect cells using sphingosine and [gamma-(32)P]-ATP at 10 uM by scintillation proximity counting
Activity at mouse SPHK2 transfected in mouse A20/2J cell lysates assessed as compound phosphorylation at 10 uM after 30 mins by autoradiography in presence of [gamma-32P]ATP
Inhibition of mouse SPHK2 in mouse A20/2J cells using [3-3H]sphingosine as substrate assessed as [3H]S1P formation at 2 to 4 uM incubated for 5 mins prior to substrate addition measured after 5 mins
Inhibition of mouse SPHK2 in mouse A20/2J cells using [3-3H]sphingosine as substrate assessed as [3H]S1P formation at 8 uM incubated for 5 mins prior to substrate addition measured after 5 mins
Inhibition of mouse recombinant SphK2 assessed as remaining enzyme activity at 10 uM using gamma[32P]ATP incubated for 20 mins by liquid scintillation counting method
Inhibition of mouse recombinant SphK2 assessed as remaining enzyme activity at 1 uM using gamma[32P]ATP incubated for 20 mins by liquid scintillation counting method
Kinase Assay: Sphingosine kinase activity was assessed as described previously (Kharel, Y., Lee, S., Snyder, A. H., Sheasley-O'Neill, S. L., Morris, M. A., Setiady, Y., Zhu, R., Zigler, M. A., Burcin, T. L., Ley, K., Tung, K. S. K., Engelhard, V. H., Macdonald, T. L. and Lynch, K. R. Sphingosine kinase 2 is required for modulation of lymphocyte traffic by FTY720. J Biological Chemistry 280: 36865-36872 (2005)). High levels of human sphingosine kinase type 1 (mSK1) and mouse sphingosine kinase type 2 (mSK2) were expressed in Sf9 insect cells by infection with cognate baculoviruses. Crude homogenates were incubated with γ-[32P]ATP and 10 micromolar D-erythro-sphingosine in the presence of 100 micromolar concentrations of test compounds for 20 minutes at 37 C. The product, radiolabeled S1P, was isolated by extraction into organic solvents after acidification and displaced by thin layer chromatography.
Inhibition of recombinant mouse SphK2 expressed in baculovirus infected sf9 cells using D-erythro-sphingosine as substrate in presence of [gamma-33P]ATP by scintillation counting method
Inhibition of recombinant mouse SphK2 expressed in baculovirus infected Sf9 insect cells using D-erythro-sphingosine as substrate measured after 30 mins in presence of [gamma32P]-ATP by liquid scintillation counting method