Tyrosine-phosphorylated S1PR1 traffics to the ER. (A) TIRF images of WT-S1PR1, Y¹⁴³F-S1PR1, and Y¹⁴³D-S1PR1 mutants with stargazin taken at 100× magnification. Scale bars, 10 μm. (B) Quantification of the surface expression of S1PR1. n = 7–9 cells/group from experiments repeated three times. (C) Confocal images of S1PR1 and ER-mCherry taken at 63× magnification. Inset: 5× magnified image of the area in rectangle. Scale bar, 5 μm and 25 μm in insets. (D) Quantification of the percent colocalization of the S1PR1 with ER-mCherry. n = 7–10 cells/group from experiments repeated three times. Data in B and D are shown as mean ± SD. ***, P < 0.0001 compared with WT and Y¹⁴³F-S1PR1 by one-way ANOVA with two-tailed paired t test. See also Fig. S1, A and B. A.U, arbitrary units.