Tethered ligand induced signaling was assessed in HEK293 cells 24 hours after transfection. For soluble and lipidated peptides, 20 hours following transfection, cells were stimulated in serum-free medium for an additional 4 hours. The activity of all experimental ligands (MTLs and SMALs) was compared to a 4 hour treatment of receptors with amidated endogenous ligands: s-SubP-NH2 for NK1-3R, s-CCK4-NH2 for CCK2R, and sulfated s-CCK8-NH2 for CCK1R. For antagonist assays, CP 99994 or YM022 (Tocris, Minneapolis, MN) were added concurrently with soluble agonist for 4 hours. With tethered ligands, antagonists were added 4 hours after transfection; activity was assessed following an additional 20 hour incubation. Quantification of luciferase and β-galactosidase activities were performed as previously described [6] (link). Data were analyzed by nonlinear curve fitting using Graph Pad Prism 5.0 software.
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