FRET between U2AF35 and SF2/ASF measured by FLIM. (A) HeLa cells were cotransfected with EGFP-U2AF35 and either mCherry-C1 or mCherry-SF2/ASF. Confocal images are of transfected cells and FLIM images of the same cells, in which FRET efficiency and FRET amplitude are shown in pseudocolor. The color scale with the respective efficiency (%) is indicated. Top, EGFP-U2AF35 + mCherry-C1; Middle, EGFP-U2AF35 + mCherry-SF2/ASF; Bottom, EGFP-U2AF35 + mCherry-SF2/ASF in the presence of DRB. Bars, 10 μm. (B) FRET efficiencies determined by FLIM for interaction of SF2/ASF with U2AF35 in the presence and absence of DRB. Plot is of mean FRET efficiencies ± SD for seven to nine cells. To measure the FRET efficiency in the speckles and nucleoplasm, a region characteristic of each was selected for each cell. P-values were obtained as described in the Fig. 1 legend. *, P < 0.1.