The NFAT-reporter assay

The nuclear factor of activated T-cells response element (NFAT-RE) reporter assay was performed according to the luciferase reporter assay for deciphering GPCR pathways paper²³ and Promega instruction. Briefly, AD293 cells were split into 24 well plates at a density of 40,000 per well. After one day of growth on 37 °C at 5% CO₂, cells (per well) were transfected with 100 ng of NFAT-RE-Luc, 10 ng of pcDNA3-H1R wild-type or mutations, 10 ng of phRGtkRenilla plasmids by X-tremeGENE HP (Roche) at a ratio 3:1 to DNA amount. 16 h after transfection, cells were induced by histamine at 10 µM or vehicle. Six hours after induction, cells were harvested and lysed by addition of 1× Passive Lysis Buffer (Promega), and luciferase activity was assessed by the Dual-Glo Luciferase system (Promega). Data were plotted as firefly luciferase activity normalized to Renilla luciferase activity in Relative Luciferase Units (RLU).