![Y143D-S1PR1 augments [Ca2+]iin a Gi-dependent manner. (A) Traces showing increase in [Ca2+]i in response to 1 μM S1P from HUVEC transducing vector, GFP-WT-S1PR1, GFP-Y143F-, or GFP-Y143D-S1PR1 mutants. (B) Mean ± SD of [Ca2+]i from 6–10 cells in a field from experiments repeated multiple times. Basal Ca2+ (−) analyzed at 45 s; peak increase in Ca2+ analyzed at 90 s. ***, P < 0.0001 compared with unstimulated vector or WT-S1PR1; ###, P < 0.0001 compared with S1P-stimulated WT-S1PR1 by one-way ANOVA with two-tailed paired t test. (C) [Ca2+]i (+) in response to 1 μM S1P from ECs expressing cDNA as in A after pretreatment with PTX (50 μM) for 2 h. (D) Mean ± SD of [Ca2+]i from 6–10 cells in a field from experiments repeated multiple times. Basal Ca2+ (−) analyzed at 45 s; peak increase in Ca2+ analyzed at 90 s. ***, P < 0.0001 compared with unstimulated vector or WT-S1PR1; ###, P < 0.0001 compared with S1P-stimulated WT-S1PR1 by one-way ANOVA with two-tailed paired t test. (E) Pulldown of Gi with S1PR1 from ECs expressing vector or S1PR1 cDNA as in A. Anti-GFP antibody was used for pulldown. Immunocomplexes were probed using anti-Giα antibody. β-Actin was used as a loading control. (F) Densitometry of Giα normalized against β-actin. Experiments were repeated three times independently. (G) ER Ca2+ release versus SOCE from ECs transducing cDNAs as in A. ECs bathed in Ca2+-free medium were stimulated with 1 μM S1P to determine Ca2+ release (first peak). After Ca2+ declined to basal level, 1.5 mM Ca2+ was readded to induce SOCE. (H) Mean ± SD of [Ca2+]i (n = 7–10 cells/group) from experiments repeated three times. Basal Ca2+ (−) was analyzed at 20 s; peak increase in Ca2+ from the ER was analyzed at 45 s, while peak of SOCE was analyzed 210 s. *, P < 0.05; ***, P < 0.0001 compared with unstimulated vector or WT-S1PR1; ###, P < 0.0001 compared with WT-S1PR1 after S1P stimulation by one-way ANOVA with two-tailed paired t test. See also Fig. S4, A and B. IB, immunoblot; IP, immunoprecipitation; UD, undetectable. (E) Molecular weight marker in kD.](https://cdn.rupress.org/rup/content_public/journal/jcb/220/12/10.1083_jcb.202006021/3/jcb_202006021_fig6.png?Expires=1767699694&Signature=lfkHyW2IX2HOKABYlf-WxZwi7884SkBE3ZPA-tpZkKJmsDiVQc5x82e3pLz74JR3iLVYnObAX1RrCqrPA0iioczJ0wyrriLVWf5AsMut68~WkKst-YcZClhCRjAgHGekoIhq8C7Z7th0mO6KThcHz4NbjGV-1ShkDJHWSzkS5JelvuSBpMaIKQW1W186mDj~bciyFO3LgqENQepZeW3FjL2gkBum3zWRp0Jbvfd-bDCDn6TysP0duHIPLVDnh1~1lfWA-vlm2hBXjYcv~jkeyElxARPW6f2TaBBWNWB4A2SFavfZL5jc6Cmp5WGkH5veNLqeAVM4Khf9U3LkEU~vmw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Y143D-S1PR1 augments [Ca2+ ] i in a Gi-dependent manner. (A) Traces showing increase in [Ca2+]i in response to 1 μM S1P from HUVEC transducing vector, GFP-WT-S1PR1, GFP-Y143F-, or GFP-Y143D-S1PR1 mutants. (B) Mean ± SD of [Ca2+]i from 6–10 cells in a field from experiments repeated multiple times. Basal Ca2+ (−) analyzed at 45 s; peak increase in Ca2+ analyzed at 90 s. ***, P < 0.0001 compared with unstimulated vector or WT-S1PR1; ###, P < 0.0001 compared with S1P-stimulated WT-S1PR1 by one-way ANOVA with two-tailed paired t test. (C) [Ca2+]i (+) in response to 1 μM S1P from ECs expressing cDNA as in A after pretreatment with PTX (50 μM) for 2 h. (D) Mean ± SD of [Ca2+]i from 6–10 cells in a field from experiments repeated multiple times. Basal Ca2+ (−) analyzed at 45 s; peak increase in Ca2+ analyzed at 90 s. ***, P < 0.0001 compared with unstimulated vector or WT-S1PR1; ###, P < 0.0001 compared with S1P-stimulated WT-S1PR1 by one-way ANOVA with two-tailed paired t test. (E) Pulldown of Gi with S1PR1 from ECs expressing vector or S1PR1 cDNA as in A. Anti-GFP antibody was used for pulldown. Immunocomplexes were probed using anti-Giα antibody. β-Actin was used as a loading control. (F) Densitometry of Giα normalized against β-actin. Experiments were repeated three times independently. (G) ER Ca2+ release versus SOCE from ECs transducing cDNAs as in A. ECs bathed in Ca2+-free medium were stimulated with 1 μM S1P to determine Ca2+ release (first peak). After Ca2+ declined to basal level, 1.5 mM Ca2+ was readded to induce SOCE. (H) Mean ± SD of [Ca2+]i (n = 7–10 cells/group) from experiments repeated three times. Basal Ca2+ (−) was analyzed at 20 s; peak increase in Ca2+ from the ER was analyzed at 45 s, while peak of SOCE was analyzed 210 s. *, P < 0.05; ***, P < 0.0001 compared with unstimulated vector or WT-S1PR1; ###, P < 0.0001 compared with WT-S1PR1 after S1P stimulation by one-way ANOVA with two-tailed paired t test. See also Fig. S4, A and B. IB, immunoblot; IP, immunoprecipitation; UD, undetectable. (E) Molecular weight marker in kD.