Fig. 3. Determination of FRET between TRPV channel subunits. (A-D) HEK293 cells were transiently co-transfected with expression plasmids encoding CFP- or YFP-fused TRPV channels as indicated. The relative CFP and YFP fluorescence intensities in single cells expressing the respective TRPV construct were determined and averaged ([CFP]_r and [YFP]_r) to ensure comparable TRPV expression. FRET efficiencies were determined by measuring the recovery of CFP fluorescence during YFP photobleaching. Cells were excited at 410 nm and 515 nm for CFP and YFP detection, respectively. YFP was bleached with an illumination at 512 nm for 2.1 seconds. FRET efficiencies between identical TRPV channel subunits are shown as grey bars. FRET experiments between different channel subunits are shown as black bars. Bars represent mean±s.e. of at least three independent experiments.