Figure 2
Effect of LUF6000 on agonist-induced activation of the human A 3 AR studied with a [35S]GTPγS binding assay. Incubations were started by addition of the membrane suspension (5 μg protein/tube) to the test tubes, and carried out in duplicate for 30 min at 25°C, except for B and C in which LUF6000 or both LUF6000 and an agonist were incubated for 20 min with membranes and other components before the addition of [35S]GTPγS (final concentration 0.2 nM). The experimental procedures are described in the Materials and Methods section. Results were from 3–5 independent experiments performed in duplicate. The basal values typically ranged from 800 to 1200 cpm. The maximal values are typically from 2000 to 2500 cpm. A. Cl-IB-MECA. B. Cl-IB-MECA (LUF6000 was incubated with membranes 20 min before the addition of other components). C. Cl-IB-MECA (Both LUF6000 and Cl-IB-MECA were incubated 20 min with membranes before the addition of other ingredients); D. NECA; E. MRS541; MRS542. The maximum stimulation of NECA in the absence of enhancers was expressed as 100%.