(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.<p><b>Copyright information:</b></p><p>Taken from "Spatial mapping of splicing factor complexes involved in exon and intron definition"</p><p></p><p>The Journal of Cell Biology 2008;181(6):921-934.</p><p>Published online 16 Jun 2008</p><p>PMCID:PMC2426932.</p><p></p

(A) Cell extracts prepared from 293T cells were incubated with either a mouse monoclonal anti–U1 70K antibody bound to Sepharose beads (lanes 2 and 3) or Sepharose beads alone (lanes 4 and 5)

The bound proteins were analyzed by Western blotting with anti-SF2/ASF antibody. Alternatively, the assay was performed in the presence of RNase (lanes 3 and 5). (B) In vivo detection of protein–protein interactions between ECFP-U1 70K and EYFP-SF2/ASF by FRET acceptor photobleaching microscopy. HeLa cells coexpressing ECFP-U1 70K and EYFP-SF2/ASF were analyzed on a wide-field fluorescent microscope. Images were acquired before and after photobleaching. A nonbleached region similar to the bleached region (arrows) was included in the data analysis for comparison. Bars, 15 μm. (C) Donor and acceptor mean fluorescence intensities monitored in the bleached and nonbleached regions were plotted over time. (D) FRET efficiencies for the interaction between ECFP-U1 70K and EYFP-SF2/ASF in the presence and absence of DRB. A FRET efficiency for these interactions was calculated as described in Materials and methods and, when >5%, was considered significant. Plot is of FRET efficiencies ± SD (mean for 8–27 cells) between ECFP + EYFP pairs before and after DRB treatment. P-values were obtained from the two-tailed homoscedastic test comparing the FRET efficiencies with and without DRB treatment.<p><b>Copyright information:</b></p><p>Taken from "Spatial mapping of splicing factor complexes involved in exon and intron definition"</p><p></p><p>The Journal of Cell Biology 2008;181(6):921-934.</p><p>Published online 16 Jun 2008</p><p>PMCID:PMC2426932.</p><p></p

(A) Plot of FRET efficiencies ± SD (mean for 7–14 cells) between ECFP and EYFP fusion proteins measured by FRET acceptor photobleaching

P-values were obtained from the test comparing the FRET efficiencies with and without DRB treatment. (B) FRET between U1 70K and SC35 measured by FLIM. HeLa cells were transfected with EGFP-U1 70K and cotransfected with either mCherry-C1 or mCherry-SC35. Confocal images are of transfected cells and FLIM images are 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-U1 70K + mCherry-C1; Middle, EGFP-U1 70K + mCherry-SC35; Bottom, EGFP-U1 70K + mCherry-SC35 in the presence of DRB. Arrowheads indicate high FRET within the nucleoplasm. Bars, 10 μm. (C) FRET efficiencies determined by FLIM for interaction of SC35 with U1 70K and U2AF35 in the presence and absence of DRB. Plot is of mean FRET efficiencies ± SD for 8–11 cells. P-values were obtained as described in A.<p><b>Copyright information:</b></p><p>Taken from "Spatial mapping of splicing factor complexes involved in exon and intron definition"</p><p></p><p>The Journal of Cell Biology 2008;181(6):921-934.</p><p>Published online 16 Jun 2008</p><p>PMCID:PMC2426932.</p><p></p

(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 legend. *, P < 0.1.<p><b>Copyright information:</b></p><p>Taken from "Spatial mapping of splicing factor complexes involved in exon and intron definition"</p><p></p><p>The Journal of Cell Biology 2008;181(6):921-934.</p><p>Published online 16 Jun 2008</p><p>PMCID:PMC2426932.</p><p></p

(A) HeLa cells were transfected with EGFP–U1 70K and cotransfected with either mCherry-C1 or mCherry-SF2/ASF

Shown are confocal images of transfected cells and FLIM images of the same cells, in which mean fluorescence lifetime is shown in pseudocolor. The color scale with the respective lifetimes (in picoseconds [ps]) is indicated. The percentage of FRET efficiencies and FRET amplitude are shown in continuous pseudocolor. The color scale with the respective FRET efficiencies (percentage) is indicated. The FRET amplitude % represents the fraction of interacting donor molecules, also defined as the FRET population % (or concentration of FRET species). (B) FRET between U1 70K and SF2/ASF, in the presence of DRB, measured by FLIM. Experiments were performed exactly as described for A, except cells were treated with 25 μg/ml of DRB for 2 h before images were taken. Bars, 10 μm. (C) FRET efficiencies calculated from FLIM measurements for the interaction of SF2/ASF with U1 70K in the presence and absence of DRB. Plot is of mean FRET efficiencies ± SD for 9–20 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 legend. *, P < 0.1.<p><b>Copyright information:</b></p><p>Taken from "Spatial mapping of splicing factor complexes involved in exon and intron definition"</p><p></p><p>The Journal of Cell Biology 2008;181(6):921-934.</p><p>Published online 16 Jun 2008</p><p>PMCID:PMC2426932.</p><p></p

(A) Extracts prepared from 293T cells transiently transfected with either EGFP-U2AF35 and pCG-T7-HCC1

4 (lanes 3 and 4) or EGFP-U2AF35 (lanes 5 and 6) were incubated with anti-T7 antibody bound to Sepharose beads. The bound proteins were analyzed by Western blotting with anti-GFP antibody. Alternatively, the assay was performed in the presence of RNase (lanes 4 and 6). (B) U2AF65 interacts with HCC1 in cultured mammalian cells. Extracts prepared from 293T were incubated with either anti-HCC1 antibody bound to Sepharose beads (lanes 2 and 4) or Sepharose beads alone (lanes 3 and 5). The bound proteins were analyzed by Western blotting with anti-U2AF65 antibody. Alternatively, the immunoprecipitate was treated with RNase (lanes 4 and 5). (C) Effect of DRB on interactions of HCC1 with U2AF35 and U2AF65. Plot is of FRET efficiencies ± SD (mean for 8–18 cells) between ECFP and EYFP fusion proteins measured by FRET acceptor photobleaching. P-values were obtained as described in the legend.<p><b>Copyright information:</b></p><p>Taken from "Spatial mapping of splicing factor complexes involved in exon and intron definition"</p><p></p><p>The Journal of Cell Biology 2008;181(6):921-934.</p><p>Published online 16 Jun 2008</p><p>PMCID:PMC2426932.</p><p></p