Figure 2.

FRET-FLIM analysis of RTNLB6 without an interaction partner (A–D) or RTNLB6 dimerization (E–I). A and E display the raw FRET-FLIM data. In B and F, pseudocolored lifetime maps show the lifetime values for each point within the region of interest, while the distribution of lifetimes across the entire image is shown in C and G, with blue shades representing longer GFP fluorescence lifetimes than green ones. D and H display representative decay curves of a single point with an optimal single exponential fit, where χ² values from 0.9 to 1.2 were considered an excellent fit to the data points (binning factor of 2). The inset in A and I are the respective confocal images for the analysis, showing the GFP construct in green and the mRFP construct in red. This example of FRET-FLIM analysis shows that RTNLB6 homodimerizes, because the lifetime values for the GFP/mRFP fusion pair (H; 2.38 ± 0.01 ns) are lower than those for the GFP fusion alone (D; 2.63 ± 0.06 ns). Bars = 5 µm.