Figure 1 - Determination of an association between MOR and _2A-AR.

(a) Effect of photobleaching. Emission intensities of YFP (535 nm, black) and CFP (480 nm, gray) recorded from single cells coexpressing MOR^YFP and _2A-AR^CFP using fluorescence microscopy. Emission intensities were recorded before and after YFP (the acceptor fluorophore) was photobleached by 5 min exposure to light at 500 nm. (b) FRET efficiency calculated according to equation (2) from cells expressing a combination of N-terminally tagged CFP and YFP molecules (CFP^m and YFP^m), or C-terminally CFP- or YFP-tagged receptors: CFP^m/YFP^m (n = 8); PTHR^CFP/YFP^m (n = 4); YFP^m/_2A-AR^CFP (n = 4); A₁R^YFP/_2A-AR^CFP (n = 4); MOR^YFP/_2A-AR^CFP (n = 8); ^**P < 0.001. Data indicate the mean s.e.m. (c) Values of FRET efficiency from cells coexpressing MOR^YFP and _2A-AR^CFP, or CFP^m and YFP^m. Data for CFP^m and YFP^m were directly proportional to the YFP emission intensity, whereas FRET efficiency values for MOR^YFP and _2A-AR^CFP followed a hyperbolic function of acceptor intensity.