The ability of CXCR3 to stimulate Gαi activity was assessed by the TGF-α–shedding assay as previously described (62). Briefly, HEK 293 cells lacking Gαq, Gαi, Gαs, and Gα12/13 were transiently transfected with plasmids encoding CXCR3, a modified TGF-α–containing alkaline phosphatase (AP-TGF-α), and either the Gαq/i1,2 assay subunit [because Gαi2 is observed to be indispensable in CXCR3 signaling in T cells (63)] or, as a negative control, the ΔC subunit (which lacks the distal amino acid residues of G protein α subunits required for receptor interaction) and were reseeded 24 hours later in HBSS (Gibco, Gaithersburg, MD) supplemented with 5 mM Hepes in a Costar 96-well plate (Corning Inc., Corning, NY). Cells were then stimulated with the indicated concentration of ligand for 1 hour. Conditioned medium (CM) containing the shed AP-TGF-α was transferred to a new 96-well plate. Both the cell and CM plates were treated with para-nitrophenylphosphate (p-NPP; 100 mM) (Sigma-Aldrich, St. Louis, MO) substrate for 1 hour, which is converted to para-nitrophenol (p-NP) by AP-TGF-α. This activity was measured at OD405 (optical density at 405 nm) in a Synergy Neo2 Hybrid Multi-Mode (BioTek) plate reader immediately after the addition of p-NPP and a 1-hour incubation. Gαi activity was calculated by first determining the amount of p-NP by absorbance through the following equation:100*(ΔOD 405 CMΔOD 405 CM+ΔOD 405 cell)where ΔOD 405 = OD 405 1 hour − OD 405 0 hour, and ΔOD 405 cell and ΔOD 405 CM represent the changes in absorbance after 1 hour in the cell and CM plates, respectively. Data were normalized to those from the vehicle-treated sample, and the nonspecific ΔC signal was subtracted from Gαi signal percentage AP-TGF-α release as follows:Gαi[AP activity(ligand  vehiclevehicle)]ΔC[AP activity(ligand  vehiclevehicle)]

Only the two highest concentrations of VUF11418 ligand (8 and 16 μM) resulted in consistent appreciable background ΔC signals, which resulted in larger errors relative to those from all other conditions (fig. S1G).

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