S1P induces BiP recruitment to the cytosol and activates its ATPase activity, which in turn binds tyrosine-phosphorylated receptor.(A) Cell fractionation from unstimulated or S1P (1 μM)-stimulated HUVECs after indicated times. (B) Mean ± SD of BiP density in cytosolic fraction, using VE-cadherin as the loading control. Fold increase in BiP/VE-cadherin density was calculated against values at time 0 (no S1P addition). ***, P < 0.0001 compared with unstimulated ECs by one-way ANOVA with two-tailed paired t test. (C) Confocal images showing BiP translocation from ECs transducing ER-mCherry without or with 1 μM S1P stimulation. Cells were stained with anti-BiP antibody and DAPI (to assess nucleus). Images taken at 63× magnification. Scale bar, 5 μm. Gray background indicates the area outside the image. (D) Mean ± SD of the BiP expression in the cytosol (4–8 cells/group) from experiments repeated multiple times. ***, P < 0.0001 compared with unstimulated ECs by one-way ANOVA with two-tailed paired t test. (E) Unstimulated or S1P (1 μM)-stimulated ECs were immunoprecipitated with anti-BiP antibody, and immunocomplexes were used to measure ATPase activity. **, P < 0.001; ***, P < 0.0001 compared with stimulated ECs at 20 min by one-way ANOVA with two-tailed paired t test. (F) Physical map of full-length BiP and deletion mutants. (G) Pulldown of S1PR1 from HUVECs cotransducing either HA-tagged vector (control), HA-full-length BiP, HA-BiP-ATPase domain, or HA-BiP-SBD domain along with Y143D-S1PR1-GFP. Anti-GFP was used for pulldown. Immunocomplexes were probed with anti-HA antibody to assess BiP expression. (H) Mean ± SD BiP interaction with S1PR1. Experiments were repeated at least three times independently. ***, P < 0.0001 compared with vector (control) by one-way ANOVA with two-tailed paired t test. (I) Mean ± SD of BiP ATPase activity in ECs transducing indicated cDNAs was determined as described in E. *, P < 0.05; ***, P < 0.0001 compared with vector by one-way ANOVA with two-tailed paired t test. ABD, ATP-binding domain (ATPase domain); A.U, arbitrary units; Cad, cadherin; FL, full-length; IP, immunoprecipitation; NBD, nucleotide-binding domain; PM, plasma membrane; UD, undetectable; WB, Western blot. (A and G) Molecular weight marker in kD.
Figure 8.

S1P induces BiP recruitment to the cytosol and activates its ATPase activity, which in turn binds tyrosine-phosphorylated receptor. (A) Cell fractionation from unstimulated or S1P (1 μM)-stimulated HUVECs after indicated times. (B) Mean ± SD of BiP density in cytosolic fraction, using VE-cadherin as the loading control. Fold increase in BiP/VE-cadherin density was calculated against values at time 0 (no S1P addition). ***, P < 0.0001 compared with unstimulated ECs by one-way ANOVA with two-tailed paired t test. (C) Confocal images showing BiP translocation from ECs transducing ER-mCherry without or with 1 μM S1P stimulation. Cells were stained with anti-BiP antibody and DAPI (to assess nucleus). Images taken at 63× magnification. Scale bar, 5 μm. Gray background indicates the area outside the image. (D) Mean ± SD of the BiP expression in the cytosol (4–8 cells/group) from experiments repeated multiple times. ***, P < 0.0001 compared with unstimulated ECs by one-way ANOVA with two-tailed paired t test. (E) Unstimulated or S1P (1 μM)-stimulated ECs were immunoprecipitated with anti-BiP antibody, and immunocomplexes were used to measure ATPase activity. **, P < 0.001; ***, P < 0.0001 compared with stimulated ECs at 20 min by one-way ANOVA with two-tailed paired t test. (F) Physical map of full-length BiP and deletion mutants. (G) Pulldown of S1PR1 from HUVECs cotransducing either HA-tagged vector (control), HA-full-length BiP, HA-BiP-ATPase domain, or HA-BiP-SBD domain along with Y143D-S1PR1-GFP. Anti-GFP was used for pulldown. Immunocomplexes were probed with anti-HA antibody to assess BiP expression. (H) Mean ± SD BiP interaction with S1PR1. Experiments were repeated at least three times independently. ***, P < 0.0001 compared with vector (control) by one-way ANOVA with two-tailed paired t test. (I) Mean ± SD of BiP ATPase activity in ECs transducing indicated cDNAs was determined as described in E. *, P < 0.05; ***, P < 0.0001 compared with vector by one-way ANOVA with two-tailed paired t test. ABD, ATP-binding domain (ATPase domain); A.U, arbitrary units; Cad, cadherin; FL, full-length; IP, immunoprecipitation; NBD, nucleotide-binding domain; PM, plasma membrane; UD, undetectable; WB, Western blot. (A and G) Molecular weight marker in kD.

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