XB-IMG-117284
Xenbase Image ID: 117284
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Figure 3. TPX2-Δ nuclei form fully functional NEs. (A) TPX2-Δ nuclei import a nuclear substrate. Mock-depleted (a–d) or TPX2-Δ (e–h) nuclear assembly reactions were supplemented with rhodamine-labeled nucleoplasmin to assay nuclear import. To distinguish import from nonspecific association of nucleoplasmin with the nuclei, some reactions (c, d, g, and h) were supplemented with the inhibitor of nuclear import, wheat germ agglutinin (WGA). Nuclei were visualized by DNA stain (a, c, e, e′, and g) or rhodamine fluorescence (b, d, f, f′, and h). (e′ and f′) Higher magnification views of e and f, respectively. (B) TPX2-Δ nuclei exclude a nonnuclear substance. Mock-depleted (a and b) or TPX2-Δ (c and d) nuclear assembly reactions were spiked with rhodamine-labeled 155-kD dextran, and a small amount of the reaction was spotted onto a coverslip. Nuclei were visualized by Hoechst staining (a and c) and the dextran by fluorescence (b and d). (C) TPX2-Δ nuclei replicate their DNA. Autoradiographs of two representative replication assays are shown. [32P]dCTP was added to control (C), mock-depleted (M), or TPX2-Δ (T) extracts supplemented with ∼100 sperm chromatin/microliter, and the reactions were incubated for 3 h at room temperature to allow DNA replication. Reactions were then stopped, separated on a 0.8% agarose gel, and visualized by autoradiography. Two types of control extracts were used, with similar results: (a) duplicate reactions were treated with the inhibitor of replication, aphidicolin (AC); (b) [32P]dCTP was added to mitotic extracts. (D) Quantitation of DNA replication in four independent experiments, normalized to the amount of label incorporated in the mock-depleted controls. Error bars indicate standard deviation. Bars: (A [a–f, g, and h] and B) 25 μm; (A, e' and f') 10 μm. Image published in: O'Brien LL and Wiese C (2006) Copyright © 2006, The Rockefeller University Press. Creative Commons Attribution-NonCommercial-ShareAlike license Larger Image Printer Friendly View |