2.3. GTPγS assay

Membrane preparations of Chinese Hamster Ovary (CHO) cells, expressing recombinant human S1P₁, S1P₂, S1P₃, S1P₄, and S1P₅ receptor, or of Human Umbilical Vein Endothelial Cells (HUVEC) (Cascade Biologics Inc., UK) were used for (³⁵S)‐GTPγS‐binding assays to measure intracellular G protein activation following agonist binding. The assays were essentially performed as described previously.17 EC₅₀ values were calculated with the IC₅₀Witch software (Actelion Pharmaceuticals Ltd, Switzerland), using the maximal response of the compound as the maximum (Smart Max) and the no‐effect level of the lowest compound concentration as the minimum. The maximal effect (E _max) is expressed as a percentage of the maximal response that was induced by S1P in the same assay (either 1 μmol·L^‐1 or 10 μmol·L^‐1, depending on the S1P receptor subtype).