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Cells were cultured on collagen-coated coverslips (cat #08-115 Millipore), transfected with S1P receptor-enhanced green fluorescence protein (EGFP) fusion constructs using Lipofectamine 3000 reagent (cat #L3000001, Thermo Fisher), and serum-starved for 4 hours prior to ligand stimulation essentially as described50.
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