FIGURE 2.

Förster resonance energy transfer (FRET) between GFP- and RFP -labeled P2XR subunits measured with donor recovery by acceptor photobleaching. Measurements were made at the oocyte pole adjacent to the bottom of the recording chamber (P) or at the equator (E) of the oocytes, as shown in Figure 1. The degree of donor recovery was plotted against the degree of acceptor bleaching. FRET efficiency (FE) was obtained by extrapolating the linear regression line to complete acceptor bleaching (right hand y-axis). (A) Negative control: coexpression of C-terminally RFP-labeled hP2X4 with SP-GFP-GLYRA1. (B) In oocytes expressing either hP2X4-GFP or hP2X4-RFP, the mean changes in the donor (donor recovery) and acceptor fluorescence (acceptor bleach) were measured at bleached versus non-bleached areas. The decrease of the donor fluorescence in regions of acceptor bleach indicates a bleaching effect of the acceptor excitation light and leads to calculation of negative FE values. (C) Positive control: P2X4 protein with a C-terminal tandem GFP-RFP label. (D) Coexpression of C-terminally GFP- and RFP-labeled hP2X4 constructs. Mean fluorescence values were calculated from 50–60 regions of interests (ROIs) in 5–10 oocytes.