Dynamin internalizes tyrosine-phosphorylated S1PR1. (A) S1PR1 surface expression without (−) or with (+) dynasore (100 μM) determined using anti-S1PR1 antibody. ECs were first transfected with indicated cDNAs. Dynasore was added within 30 min after transfection, and cells were lysed after 24 h. Total S1PR1 and dynamin expression were quantified using anti-S1PR1 or anti-dynamin antibodies. (B) Densitometry of S1PR1 surface expression over total S1PR1 using blots in A. n = 3 independent experiments. (C) S1PR1 surface expression in ECs transfected with control siRNA (−) or dynamin siRNA (+) determined as in A. (D) Densitometry quantified as in B. n = 3 independent experiments. (E) Confocal images showing S1PR1 cell surface localization following cotransfection with mCherry-tagged GTPase-defective dynamin (K44A-dynamin). Scale bar, 5 μm. (F) Quantification of S1PR1 cell surface expression. n = 5–8 cells/group from experiments repeated three times. All data in B, D, and F are shown as mean ± SD. One-way ANOVA with paired two tailed t test. ***, P < 0.0001 compared with WT-S1PR1 or Y¹⁴³F-S1PR1. See also Fig. S2, A–D. A.U, arbitrary units; IB, immunoblot; IP, immunoprecipitation. (A and C) Molecular weight marker in kD.