Figure 4. Effects of S1P, FTYp, and PMA on S1P₁ receptor–Rab protein interaction.

HEK 293 cells were co-transfected with plasmids for transient expression of mCherry-tagged S1P₁ receptors and eGFP-tagged Rab proteins as described under ‘Experimental procedures’ section, and FRET was used as an index of protein–protein interaction. Cells were challenged for the times indicated with 1 µM S1P ((A) [Rab5], (D) [Rab9], and (G) [Rab7]), 10 µM FTYp ((B) [Rab5], (E) [Rab9], and (H) [Rab7]), or 1 µM PMA ((C) [Rab5], (F) [Rab9], and (I) [Rab7]). FRET values were determined and normalized to those observed in the absence of stimulus (Time 0, Baseline; 100%). Plotted are the means with vertical lines indicating the S.E.M of 24–30 samples resulting from four or five independent experiments, in which six different images were obtained in each of these. *P <0.005 compared with Baseline, **P <0.01 compared with Baseline, ***P <0.05 compared with Baseline.