cAMP assay

Approximately, 1.25 × 10⁵ NSCs or 0.25 × 10⁶ cortical cells were cultured in 0.5 ml medium unless otherwise specified. Cultured NSCs (DIV 4) or cortical cells (DIV 3) were treated with synaptamide for 10 min and cAMP levels were determined using cyclic AMP XP assay kit (Cell Signaling Technology, Danvers, MA) according to the manufacturer's protocol. Briefly, cAMP from the cell lysate was added to the kit to displace HRP-linked cAMP which was bound to an anti-cAMP XP Rabbit mAb immobilized on a 96-well plate. After removing excess cAMP, HRP substrate TMB was added to determine cAMP concentration colorimetrically. In some cases, cells were pretreated with 100 μM adenylyl cyclase inhibitor SQ22,536 (Sigma) or 0.4 μg ml⁻¹ IgG (Cell signaling Technology, Danvers, MA) or N-terminal targeting GPR110 antibody (Abmart) for 30 min before the stimulation with synaptamide. For evaluating synaptamide-induced cAMP production in GPR110 overexpressing cells, A549 cells were transfected with HA-tagged mGPR110 or empty vector (M45) for 24 h, and treated with 10–100 nM synaptamide for 10 min. In some cases, the transfected cells were pretreated with 0.4 μg ml⁻¹ GPR110 antibody or control IgG for 30 min before the synaptamide addition.