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Figure 2


Fig. 2. FRET detection of fully assembled Sm cores in speckles and CBs. (A) HeLa cells were transfected with plasmids to express YFP-SmB and CFP-SmD1. The YFP (acceptor) fluorescence was bleached in a region of interest. A transient (~1 second) increase in fluorescence of the donor was seen, resulting from the decrease in the intensity of acceptor fluorescence. (B) FRET efficiencies obtained in different regions of the nucleus in HeLa cells expressing YFP-SmB and CFP-SmD1 for 24 or 48 hours. No significant difference was seen in the efficiency calculated for each region using a one-way analysis of variance (ANOVA) test. The negative control of YFP-SmB and CFP-SmB gave a small negative value, probably a result of some inadvertent bleaching of the CFP donor by the 532 nm laser. A positive control using a single vector with YFP and CFP (pYFPCFP) separated by seven amino acids gives a FRET efficiency of 0.43 (data not shown). Efficiencies of less than 0.05 are generally regarded as not significant.





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