Cp99994

AITC (Sigma) was dissolved in dimethyl sulfoxide (DMSO) (Sigma) to obtain 10 mM stock solution. Further dilutions were made with ECS solution to reach final concentrations of 100 μM. Capsaicin (Sigma) was dissolved in DMSO to obtain a 10 mM stock solution. Further dilutions were made with ECS or Hank’s solution to reach final concentrations of 330 or 100 nM, respectively. Penicillin-streptomycin was purchased from Gibco. D-MEM-low glucose, collagenase type XI, deoxyribonuclease I, horse serum, newborn calf serum, fetal bovine serum, poly-D-lysine, glycine, NGF, pertussis toxin (PTX), SP, HK-1 were purchased from Sigma. CP99994, AMG 9810 and HC 030031 were purchased from Tocris.

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.