XB-IMG-125039
Xenbase Image ID: 125039
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Figure 4. Nuclear functions are unaffected by Nup188–Nup93 depletion. (A) Nuclei were assembled in mock- or Nup188–Nup93-depleted extracts. After 50 min, fluorescently labeled dUTP (green in overlay) and, where indicated, aphidicolin were added. After 90 min, replication was analyzed by confocal microscopy. Membranes were stained with DiIC18 (red), and chromatin was stained with DAPI (blue). (B) Nuclei were assembled in mock-, Nup205–Nup93-, Nup188–Nup93-, and Nup98-depleted extracts. After 90 min, fluorescently labeled 2-MD (not depicted), 70-kD (middle), and 10-kD (bottom) dextrans were added. DNA was stained with DAPI, and samples were analyzed by confocal microscopy. For quantitation, only nuclei with an intact NE (as judged by the exclusion of 2-MD dextrans; >95% of nuclei in each sample) were analyzed. For mock- and Nup188–Nup93-depleted samples, seven independent experiments were analyzed (error bars show the standard deviation), and for Nup205–Nup93- and Nup98-depleted samples, two independent experiments were analyzed. (C) Nuclei were assembled as in A. After 50 min, i.e., when a closed NE had formed, importin α/β– or transportin-dependent reporter substrates containing a TEV cleavage site were added. To assay the nuclear import kinetics of the reporter, NusA-fused TEV protease was added at the indicated time points. Protease cleavage was stopped after 1 min by the addition of SDS sample buffer and boiling. Lines mark the mean of four independent experiments. (D) Nuclei were assembled as in A, and an EGFP-fused shuttling substrate was added. Nuclear export function was inhibited by the addition of 300 nM leptomycin B. Nuclei were isolated and analyzed by confocal microscopy. Membranes are stained with DiIC18 (red). Bars, 20 µm. Image published in: Theerthagiri G et al. (2010) © 2010 Theerthagiri et al. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike license Larger Image Printer Friendly View |