Effect of prolonged overexpression of SphK on NF-κB activation and S1P synthesis. A. Control cells and cells overexpressing SphK (hSK) were pretreated ± 50 ng/ml pertussis toxin for 16 h before stimulation with vehicle or 3 μM S1P for 30 minutes. Proteins were extracted and NF-κB (p65)-activation was assayed by ELISA. The inset shows Western blots of 10 μg of protein from mock transduced or SphK1 overexpressing HeLa cells. The cell extracts were probed for sphingosine kinase and S1P2. B. [3H]S1P was extracted from the mock- or SphK transduced cells following a 10-minute incubation with [3H]sphingosine, and was analyzed using thin layer chromatography and scintillation counting. C. The SphK overexpressing cells were treated with 50 ng/ml pertussis toxin or vehicle for 16 h. Secreted [3H]S1P was extracted from the medium following a 10-minute incubation with [3H]sphingosine. The formed [3H]S1P was assayed using thin layer chromatography and scintillation counting. The bars in panels A-C denote the mean ± SEM of at least three independent experiments (*, p < 0.05).
Blom et al. BMC Cell Biology 2010 11:45 doi:10.1186/1471-2121-11-45