12 hht

The heptdadecanoid 12–HHT and the eicosanoid LTB4 were obtained from Cayman Chemical, Ann Arbor, USA, as ethanolic solutions. The ethanol was extensively evaporated under vacuum. Then the eicosanoids in excess respectively to the receptor were directly dissolved by the NMR sample containing the receptor associated with perDAPol in a 100%-D₂O solution (20 mM Tris/HCl buffer pH 8, 100 mM NaCl) at final concentrations of ~120 and ~140 μM of 12–HHT and LTB4, respectively. The receptor concentration was ~15 μM which gives rise to ligand/BLT2 molar ratio of 8 and ~9 for 12–HHT/BLT2 and LTB4/BLT2. Considering a percentage of properly folded receptor ranging from 50 to 70%^{49 (link)}, this means an effective ligand/receptor ratios of 15 ± 4. Synthesis of perDAPol was performed as already described^{50 (link)} and the overexpression, purification and folding of perdeuterated human BLT2 receptor is detailed in Catoire et al.^{18 (link)}.

LTB₄ and 12-HHT were purchased from Cayman Chemical (Ann Arbor, MI). Probenecid was purchased from Sigma-Aldrich (St. Louis, MO). Pluronic F-127, Alexa-488-conjugated anti-Rat IgG (Molecular Probes), and fetal calf serum (FCS; GIBCO) were obtained from Invitrogen (Carlsbad, CA). A penicillin-streptomycin solution and geneticin (G418) were purchased from Nacalai Tesque (Kyoto, Japan). [³⁵S]-guanosine 5’-O-(gamma-thio) triphosphate (GTPγS) was obtained from Perkin-Elmer Life Science (Boston, MA). Anti-hemagglutinin (anti-HA; clone 3F10) was purchased from Roche (Penzberg, Germany).