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Fig. 4. UAP56 and SRm160 are present in the same macromolecular complexes at RNA-splicing speckled domains. FRET between EGFP-UAP5 wt or UAP56 K95N (green) and mRFP-SRm160 (red) was measured in transiently transfected HeLa cells by the method of sensitized emission (Gordon et al., 1998). (A) Cells expressing both proteins and those expressing only EGFP-UAP56 or only mRFP-SRm160 were trypsinized and replated on coverslips together. Fields that contained at least one co-transfected cell and one each of the control singly transfected cells were selected for analysis (left panel). FRET was found only in cells expressing both EGFP-UAP56 and mRFP-SRm160; within these cells, FRET was found only in the regions of RNA-splicing speckled domains. FRET efficiency was calculated using Leica confocal software. The maximum FRET efficiency between mRFP-SRm160 and EGFP-UAP56 wt at speckled domains was 8%. FRET efficiencies are presented in a color-coded format with a scale to the right of the right panels. (B) There was no FRET observed in RNA-splicing speckled domains between mRFP-SRm160 and EGFP-UAP56 K95N. The maximum FRET efficiency between SRm160 and UAP56 K95N was 6%, but this was in the nucleoplasm. FRET efficiencies are presented in a color-coded format with a scale to the right of the middle panel. Scale bars: 10 µm.
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