FRET between HCC1 and both subunits of the U2AF heterodimer measured by FLIM. (A) HeLa cells were cotransfected with EGFP-HCC1 and either mCherry-C1 or mCherry-U2AF35. Confocal images are of transfected cells and FLIM images are of the same cells. The color scale with the respective efficiency (%) is indicated. The FRET efficiencies are shown in continuous pseudocolor. Top, EGFP-HCC1 + mCherry-C1; Middle, EGFP-HCC1 + mCherry-U2AF35; Bottom, EGFP-HCC1 + mCherry-U2AF35 in the presence of DRB. Arrows indicate high FRET within the nucleoplasm and arrowheads indicate nuclear speckles. (B) FRET between HCC1 and U2AF65 measured by FLIM. HeLa cells were transfected with EGFP-HCC1 and cotransfected with either mCherry-C1 or mCherry-U2AF65. Confocal images are of transfected cells and FLIM images are of same cells, in which the percentage of FRET Efficiency and FRET amplitude are shown in pseudocolor. The color scale with the respective efficiency (%) is indicated. Top, EGFP-HCC1 + mCherry-C1; Middle, EGFP-HCC1 + mCherry-U2AF65; Bottom, EGFP-HCC1 + mCherry-U2AF65 in the presence of DRB. Arrowheads indicate high FRET within the nucleoplasm and arrowheads indicate nuclear speckles. Bars, 10 μm.