cAMP accumulation and β-arrestin translocation assays

A cAMP accumulation assay was conducted using Euroscreen FAST in accordance with the service supplier's instructions. CHO-K1 cells expressing human A₃AR were mixed with forskolin and increasing concentrations of FM101. After incubating the cells with lysis buffer, cAMP concentrations were estimated using a homogeneous time-resolved fluorescence (HTRF) assay kit (Perkin Elmer, USA).

The translocation of β-arrestin was evaluated using a PathHunter™ β-arrestin assay kit (Fremont, USA) from DiscoverX according to previously described procedures (Alnouri et al., 2015[1]; Olson and Eglen, 2007[19]). To evaluate the antagonist function of FM101, CHO-K1 cells expressing human A₃AR were pre-incubated at ten concentrations ranging from 0.05 nM to 1 μM with a three-fold increase of concentration of FM101, followed by treatment with 0.06 μM of 2‐Cl‐IB‐MECA as a reference agonist. After incubation with the assay buffer, a chemiluminescent the assay signal was generated using a commercial PathHunter Detection reagent cocktail.