Fig. 2.

FRET analysis of living HEK293 cells transiently co-transfected with human WT MC4R, the S136F mutant or CB1R. HEK293 cells were co-transfected with plasmids encoding a WT-MC4R-CFP and WT-MC4R-YFP, b WT-MC4R-CFP and the mutant MC4R-S136F-YFP or c CB1R-CFP and WT-MC4R-YFP as negative control.

If protein-protein interaction takes place, the two fluorophores CFP and YFP are in close proximity (<100 Å). In this case excitation of CFP (donor) allows energy transfer to YFP (acceptor) and emission light of a characteristic wavelength are detectable.

Here, FRET signals are detected by recording the donor recovery (CFP emission, black line) before and during acceptor bleaching (YFP emission, gray line). The selective photobleaching at 512 nm results in a decrease in YFP emission accompanied by an increase in CFP emission. An increase in CFP fluorescence during YFP-bleach is direct evidence for FRET. FRET efficiencies E were calculated from the relative increase in CFP emission and the decrease in YFP emission by linear regression. The depicted data represent means ± SEM of 4–6 single cells of one representative measurement of at least three independent transfection experiments performed in triplicates.