Dynamics of the tyrosine-phosphorylated S1PR1 to the ER. (A) Confocal images of S1PR1 and ER (mCherry) without or with S1P (1 μM). Gray background indicates the area outside the image. Scale bar, 5 μm. (B) Quantification of the surface expression. n = 5–7 cells/group from experiments repeated three times. One-way ANOVA with paired two tailed t test. **, P < 0.001; ***, P < 0.0001 compared with WT-S1PR1 at 0 and 20 min and Y¹⁴³F-S1PR1 at 0, 2.5, 5, and 20 min. (C) Time-lapse images of S1PR1–Dendra2 before and after photoconversion at time 0 (indicated by green, rectangles) and after the addition of S1P (1 μM; red, rectangles). 488-nm channel (green) = unconverted S1PR1; 561-nm channel (red) = photoconverted S1PR1. Scale bar, 10 μm. (D and E) Quantification using images in C. n = 5–6 cells from experiments repeated two times. (D and E) One-way ANOVA with paired two tailed t test. ***, P < 0.0001 compared with WT-S1PR1-Dendra2 at basal levels or after S1P stimulation at 2.5 min (D) or at 1.5 min (E). Data in B, D, and E are shown as mean ± SD. See also Fig. S1, C and D. A.U, arbitrary units; UD, undetectable.