Whole Cell Competition Binding Assays

Radioligand competition binding was performed as previously described^10,11 on whole cells seeded into 96-well plates and cultured overnight. For homologus competition binding experiments, cells were incubated overnight at 4 °C with ∼100 pM ¹²⁵I-sCT_(8–32) (specific activity, 2000 Ci/mmol) and serial dilutions of noniodinated sCT_(8–32). For heterologous competition binding assays, cells were incubated overnight with the antagonist radioligand ¹²⁵I-sCT_(8–32), and serial dilutions of competing unlabeled peptide. Nonbound ligand was removed and bound ligand activity was measured using a γ counter (Wallac Wizard 1470 Gamma Counter, PerkinElmer, 78% counter efficiency). Values were normalized against nonspecific binding, defined by the presence of 1 μM of unlabeled sCT_(8–32), and total ligand bound radioligand.