TGF-α–shedding assay

The ability of CXCR3 to stimulate Gα_i activity was assessed by the TGF-α–shedding assay as previously described (63). Briefly, HEK 293 cells lacking Gα_q, Gα_i, Gα_s, and Gα_12/13 were transiently transfected with plasmids encoding CXCR3, a modified TGF–α-containing alkaline phosphatase (AP-TGF-α), and either the Gα_q/i1,2 assay subunit [because Gα_i2 is observed to be indispensable in CXCR3 signaling in T cells (64)] or, as a negative control, the ΔC subunit (which lacks the distal amino acid residues of G protein α-subunits required for recptor interaction) and were reseeded twenty-four hours later in Hanks’ Balanced Salt Solution (HBSS) (Gibco, Gaithersburg, MD) supplemented with 5mM 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 one hour, which is converted to para-nitrophenol (p-NP) by AP-TGF-α. This activity was measured at OD₄₀₅ in a Synergy Neo2 Hybrid Multi-Mode (BioTek) plate reader immediately after the addition of p-NPP addition and a 1-hour incubation. Gα_i activity was calculated by first determining the amount of p-NP by absorbance through the following equation:

where ΔOD 405=OD 405 1 hour− OD405 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 non-specific ΔC signal was subtracted from Gα_i signal percentage AP-TGF-α release as follows:

Only the two highest concentrations of {"type":"entrez-protein","attrs":{"text":"VUF11418","term_id":"1711671772","term_text":"VUF11418"}}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).