cAMP accumulation measurement

After seeding in a 96-well plate overnight, HEK293 cells stably cotransfected with Flag-Aj5-HT4R and pCRE-Luc vectors were grown to 80–85% confluence, before being starved for 24 h in serum-free medium to eliminate the effects of the endogenous 5-HT in FBS. Cells were thentreated with the indicated concentration of 5-HT(1 pM, 10 pM, 100 pM, 1 nM, 10 nM, 100 nM, 1 mM, and 10 mM) in DMEM without FBS and incubated for 4 h at 37 °C, with application repeated a total of three times. Luciferase activity was detected using a firefly luciferase assay kit (Kenreal, Shanghai, China).