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Fig. 2. Both FRET and immunoprecipitation reveal a close molecular association of Tip60 and AICD in AFT-complexes. (A) Two cells with nuclear AFT-complexes. Sensitized emission of citrine after excitation of cyan fluorescent protein (CFP) was calculated from raw FRET values with correction for CFP bleach-through and citrine cross-excitation. To prevent quenching of citrine emission, Fe65 was labeled with a Cy5-conjugated antibody (data not shown). (B) Control cells co-expressing CFP and citrine targeted to the nucleus with nuclear localization signals showed very little sensitized emission of citrine. (C) FRET measurements of AFT-complex-containing spherical nuclear spots showed sensitized emission from citrine-AICD in the absence of the Cy5-conjugated antibodies against Fe65. (D) Citrine-AICD was bleached in one nucleus and the intensity of CFP measured in single spots before (pre) and after bleaching (post). The graphs show the mean pixel intensity of the measured CFP-fluorescence. The pre/post values of single spots are connected by a line. After bleaching, CFP fluorescence intensities increased in spots in the bleached nucleus, but not in surrounding spots in unbleached nuclei. Bar, 10 µm. (E) Immunoprecipitation (IP) of AFT-complex components from nuclear fractions. Antibodies against AICD or Tip60 co-precipitate Fe65 as detected by western blot (WB) of the HA tag. IP with antibodies against AICD precipitated Tip60 and, vice versa, antibodies against Tip60 precipitated AICD, as shown by staining with an antibody against fluorescent proteins that detects both CFP and citrine. The lower band in panel 4 is unspecific, and results from the detection of the antibody used for immunoprecipitation by the secondary antibody used in western blots. Marker proteins are 19, 28, 39, 51, 64, 97 and 191 kDa.
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