(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>