2.6. ERK1/2 phosphorylation assay

CHO-hA_2AR cells [21] were seeded in 96-well plates (Greiner bio-one; 2.5 × 10⁴ cells/well) in the absence of G418 20 h prior to assay. The medium was replaced with medium lacking serum and incubated for an additional 3 h. Cells were either pretreated or left untreated (control) prior to stimulation. For the pretreatment, CHO-hA_2AR cells were washed twice with HBSS and incubated in HBSS containing ADA (3 U/ml) and adenosine 5′-[α,β-methylene] diphosphate (50 μM) for 15 min at 37 °C. Control as well as pretreated cells were washed with warm HBSS to remove ADA and incubated with adenosine 5′-[α,β-methylene] diphosphate (50 μM), inosine and AEA061 at indicated concentration(s) in the presence or in the absence of ZM 241385 (100 nM) for 10 min at 37 °C. Pretreated cells were also incubated with the same assay components in the presence of ADA (3 U/ml) for 10 min at 37 °C. The assay was terminated by aspirating the assay buffer and incubating cells with lysis buffer (50 μl/well) at room temperature with shaking for 10 min. Phospho ERK1/2 levels were detected using an Alphascreen Surefire kit (PerkinElmer) according to the manufacturer’s suggested protocol. Briefly, 10μl of the lysate was transferred to a ProxiPlate-384 (PerkinElmer) and incubated with 10 μl of assay detection mixture at room temperature in the dark for 2 h. Fluorescent emissions were quantified using an EnSpire multimode plate reader (PerkinElmer).