Tethered Ligand Induced Signaling in HEK293 Cells

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.