TNF-α fails to phosphorylate Y¹⁴³F-S1PR1. (A and B) HUVECs were transfected with control (siSc) or BiP siRNA (siBiP). Lysates were collected at the indicated times and probed for BiP and β-actin. A shows a representative blot image, whereas B shows the densitometric analysis of BiP depletion with β-actin as control. (C and D) HUVECs were transfected with GFP-tagged Y¹⁴³F-S1PR1 or Y¹⁴³D-S1PR1 cDNAs for 24 h, after which cells were left unstimulated or stimulated with TNF-α for 15 and 30 min in Y¹⁴³F-S1PR1–transducing ECs. Equal amounts of protein lysates were immunoprecipitated with anti-GFP antibody, and S1PR1 phosphorylation on Y¹⁴³ residue and interaction with BiP was determined in immunocomplexes using anti-phosphotyrosine and anti-BiP antibodies. Lysates were probed for total S1PR1 to assess the total protein expression and loading. C shows a representative blot from experiments that were repeated at least three times, while D shows the densitometric analysis of phosphotyrosine with S1PR1 as control. Significance was determined by one-way ANOVA followed by multiple comparisons between groups. (A and C) Molecular weight marker in kD.