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. 2020 Nov 27;3:722. doi: 10.1038/s42003-020-01451-w

Fig. 2. Pharmacological and biochemical characterisation of 1.

Fig. 2

a TR-FRET saturation binding curves obtained by treating membranes containing Lumi4-Tb labelled SNAP-A2AR with increasing concentrations of 1 in the absence (closed circles) or presence (open circles) of 10 µM ZM241385 for 1 h at 37 °C prior to determination of TR-FRET ratio. Data shown are representative of four experiments and each data point represents mean ± s.e.m of triplicate determinations. b TR-FRET measurements as detailed in a taken after 1, 3 and 5 h for 4 nM (open circles) and 250 nM (diamonds) 1. Data shown  are specific TR-FRET ratios and represents mean ± SEM of four experiments performed in triplicate. c Membranes containing Lumi4-Tb labelled SNAP-A2AR were pre-treated with 250 nM 1 (5 h, black circles) or CA200645 (2 h, green squares) at 37 °C prior to measurement of TR-FRET ratio. After basal reads, 10 µM ZM241385 was added and measurements taken every 2 min (1) or 5 s (CA200645) for 5 min (CA200645) or 180 min (1). Non-specific binding was determined in the presence of 10 µM ZM241385 and data normalised to total and non-specific binding at the zero time point. Each data point represents mean ± s.e.m. of four experiments each performed in triplicate. d T-RexTM-293 cells induced to express TS-SNAP-A2AR were treated with 500 nM 1 in the presence or absence of 1 µM ZM241385. Untreated cells were used as a control. TS-SNAP-A2AR was purified and separated on an SDS-PAGE gel and direct Cy5 fluorescence was visualised using in-gel fluorescence. Gel shown is representative of three independent experiments. e CHO CRE-SPAP cells were treated with (open squares) or without (closed circles) 1 µM 1 for 16 h. Cells were washed for 30 min prior to the addition of increasing concentrations of CGS21680 and levels of CRE-mediated SPAP production measured after 5 h. Data are normalised to basal (in the absence of agonist and 1) and maximal CGS21680 response in the absence of 1 and each point represents the mean ± s.e.m. of five experiments performed in triplicate.