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Fig. 5. NoLS still colocalizes with B23 after actinomycin D or roscovitine treatment and is delocalized from nucleoli to nucleoplasm when the endogenous B23 is selectively delocalized from nucleoli to nucleoplasm. (A) HeLa cells cotransfected with GFP-NoLS and mDsRed-B23 or with GFP-NoLS and fibrillarin-RFP were treated with actinomycin D (a-h), or roscovitine (i-p). After actinomycin D treatment, NoLS colocalized with B23 in the segregated nucleolus (a-d) but was excluded from the fibrillarin-containing caps (e-h). After roscovitine treatment, NoLS colocalized with B23 in large nuclear bodies (i-l) but not with fibrillarin (m-p). (B) NIH-3T3 cells were transfected with GFP-B23-NES and analyzed by B23 immunolabeling (a-c) or cotransfected with GFP-B23-NES and DsRed-NoLS (d-f). After leptomycin B treatment, GFP-B23-NES was located in the nucleus and mostly excluded from nucleoli (a, arrowheads). Anti-B23 antibodies, revealing both endogenous and mutated B23, labeled the nucleoplasm and only slightly the nucleoli of GFP-B23-NES-expressing cells, in contrast to what was observed in cells not expressing GFP-B23-NES in which B23 labeling was mostly restricted to nucleoli (b). Thus, the endogenous B23 was mostly excluded from nucleoli in NIH-3T3 cells expressing GFP-B23-NES. In cells expressing GFP-B23-NES (d), DsRed-NoLS (e) was also mostly delocalized from nucleoli to nucleoplasm. Arrowheads point to nucleoli. Bars, 10 µm.
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