Figure 3.

The S1P₅ receptor displays ligand-independent inhibition of ERK activity. (A) CHO cells were cotransfected with human S1P₅ receptor cDNA or vector (mock) and HA-ERK2 cDNA. After incubation in serum-free medium for 24 h, cells were stimulated with medium containing 10% FBS with or without addition of 1 μM S1P. After cell lysis, immunoprecipitation, in vitro kinase reactions and separation by SDS page (see Methods), myelin basic protein (MBP) phosphorylation was quantified for measurement of ERK activity. HA-ERK2 was quantified via Western blot for confirmation of equal expression levels. (B) The assay was essentially carried out as described above but stimulation was performed in medium containing 10% csFBS with or without S1P. Data shown are representative for 2–4 independent experiments.