S1PR1 internalizes independent of Rab-GTPases. (A and B) HUVECs were transfected with control siRNA (siSc) or siRNA against Rab5 (siRab5) or Rab7 (siRab7) GTPases. After 24 h, cells were again cotransduced with WT-S1PR1-GFP and ER-mCherry. 48 h after transfection, cells were stimulated with S1P at the indicated time points, fixed, and stained with anti-Rab5 and anti-Rab7 antibodies followed by DAPI (nuclear stain). A shows the representative images (scale bar, 5 μm), whereas B shows the mean ± SD quantification of the surface intensity of S1PR1-GFP in HUVECs transfected with siRab5 and siRab7. n = 4–6 cells from experiments that were repeated at least two times. Gray background indicates the area outside the image. **, P < 0.001; ***, P < 0.0001 compared with S1P-stimulated siSc or siRab5 or siRab7 ECs transfected with S1PR1-GFP at 2.5 min by one-way ANOVA with two-tailed paired t test. (C and D) At 48 h after transfection, lysates from control or Rab5/Rab7-depleted ECs were immunoblotted using anti-Rab5 and anti-Rab7 antibodies with β-actin as a loading control. Mean ± SD Rab5 and Rab7 densities were quantified against β-actin. Blot represents experiments that were repeated multiple times independently. ***, P < 0.0001 compared with siSc-transfected ECs by one-way ANOVA with two-tailed paired t test. A.U, arbitrary units. (C) Molecular weight marker in kD.