Phosphorylated S1PR1 interacts with BiP. (A) Pulldown of BiP from HUVEC transducing vector, GFP-S1PR1, or GFP-Y¹⁴³D-S1PR1 after S1P stimulation at indicated times. Anti-GFP antibody was used to pull down; immunocomplexes were probed with anti-BiP antibody to assess interaction. Anti-GFP or anti-β-actin antibody was used to confirm total protein. (B) Mean ± SD of BiP interaction with S1PR1 using blots in A and with β-actin as loading control. n = 3 independent experiments. ***, P < 0.0001 compared with unstimulated WT-S1PR1 by one-way ANOVA with two-tailed paired t test. (C) Pulldown of BiP from ECs expressing GFP-Y¹⁴³F- or GFP-Y¹⁴³D-S1PR1 mutants processed as in A. (D) Mean ± SD of the BiP interaction with S1PR1 normalized using total S1PR1. n = 3 independent experiments. (E) Confocal images illustrating colocalization of S1PR1 with BiP without or with S1P stimulation. Cells were also stained for DAPI to assess nuclei. Gray background indicates the area outside the image. Scale bar, 5 μm. (F) Quantitation of the colocalization index plotted as mean ± SD. ***, P < 0.0001 compared with unstimulated WT-S1PR1 or Y¹⁴³F-S1PR1 by one-way ANOVA with two-tailed paired t test. See also Fig. S3, A–E. A.U., arbitrary units; IB, immunoblot; IP, immunoprecipitation; UD, undetectable. (A and C) Molecular weight marker in kD.