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Fig. 3. Localization of full-length and mutant NRF in interphase nuclei. (A) Fluorescence microscopy images (b,d) and the corresponding phase-contrast images (a,c) of C243 cells expressing His-Myc-tagged NRF (a,b) or NRF-GFP fusion protein (c,d). To visualize the cytoplasmic and nucleoplasmic presence of NRF, confocal laser scanning microscopy of GFP-tagged full-length protein was carried out under non-saturating (e) and saturating (f) imaging conditions in living C243 cells. (B) C243 cells expressing full-length NRF-GFP (a), NRF^362-690-GFP (b) or the nucleolic marker protein GFP-hfbr2_82i24 (d) were treated with 5 µg/ml actinomycin D. Intracellular localization of the GFP-tagged proteins was determined by confocal laser scanning microscopy in living cells. Representative images from cells after 8 hours of actinomycin D treatment are shown. As non-treated control GFP-hfbr2_82i24 is shown in c. (C) C243 cells expressing His-Myc-tagged NRF were either left untreated or treated with 5 µg/ml actinomycin D for 8 hours. Nuclear and nucleolar fractions were prepared as described under Materials and Methods. Equal amounts of protein were resolved by SDS-PAGE, blotted onto a nitrocellulose membrane and probed with antibody directed against the Myc-tag. (D) NRF deletion mutants fused to GFP were expressed in C243 cells. The subcellular localization of the various mutant proteins was determined by confocal laser scanning microscopy in living cells. (a) NRF^1-550-GFP, (b) NRF^1-480-GFP, (c) NRF^1-434-GFP, (d) NRF^1-402-GFP, (e) NRF ^1-361-GFP, (f) NRF^362-690-GFP.

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