Phosphorylated S1PR1 internalizes endogenous S1PR1. (A) Confocal image at 63× magnification from HUVECs coexpressing GFP-WT-S1PR1 and ER-mCherry along with HA-tagged vector or HA-Y¹⁴³F- or HA-Y¹⁴³D-S1PR1 mutants. Scale bar, 5 μm. (B) Quantification of the S1PR1 surface intensity using images in A. n = 5–6 cells/group from experiments repeated two times. (C) TEER trace in ECs coexpressing WT-S1PR1-GFP and HA-tagged vector or S1PR1 mutants. (D) Mean ± SD of TEER using multiple traces in C. Basal TEER (−) = values between 10 and 30 min; S1P-stimulated TEER (+) = values between 2 and 2.5 h. (D) One-way ANOVA with paired two tailed t test. ***, P < 0.0001 compared with unstimulated cells; ###, P < 0.0001 compared with S1P-stimulated WT-S1PR1 + vector or HA-Y¹⁴³F-S1PR1. A.U, arbitrary units; UD, undetectable.