Xenbase Image ID: 49187

Figure 4. Importin α2 and Ntf2 Are Sufficient to Account for Interspecies Nuclear Scaling by Regulating LB3 Import(A) X. tropicalis nuclei were assembled in the presence of anti-Ntf2 or IgG control antibodies (0.1 mg/ml) and 0.14 μM GFP-LB3 as indicated, and at 40 min, 0.8 μM importin α-E was added to some reactions. LB3 was visualized in nuclei by immunofluorescence at 80 min, and images for at least 50 nuclei per condition were acquired with the same exposure time. NE surface area and LB3 fluorescence were quantified. Average fold change from the buffer control and SD are shown (n = 5 extracts). Scale bar, 20 μm.(B) Wild-type and mutant GFP-LB3 proteins, 1 × Npl (nucleoplasmin), and GFP-NLS were added at 0.14 μM to X. tropicalis extract. For 5 × Npl, 0.7 μM Npl was added. Nuclei were visualized at 75 min by immunofluorescence using an antibody against Ran. NE surface area was calculated for at least 50 nuclei. Average fold change from the buffer control and SD are shown (n = 3 extracts). The K31Q mutant had a dominant-negative effect on the structure of the lamina, as nuclei were smaller and appeared crumpled, whereas the R385P mutant did not efficiently assemble into the lamina.(C) Nuclei were visualized by immunofluorescence with an antibody against Xenopus LB3. Images for at least 50 nuclei at each time point were acquired with the same exposure time. Fluorescence intensity was quantified, averaged, and normalized to 1.0 (arbitrary units). Error bars represent SD. One representative experiment of three is shown. The Western blot was performed as in Figure 2A using an antibody against Xenopus LB3.(D) Nuclei were assembled in X. tropicalis extract mock- and immunodepleted of LB3 (0.1 μM depleted). Ntf2 antibodies, importin α-E, and GFP-LB3 were added to LB3-depleted extract in the same manner as in (A) with the exception that GFP-LB3 was added at 0.2 μM. At 80 min, nuclei were stained for Ran by immunofluorescence, images for at least 50 nuclei per condition were acquired, and NE surface area was quantified. Average fold change from the mock depletion and SD are shown (n = 4 extracts). Scale bar, 20 μm.See also Figure S4 and Table S1. Image published in: Levy DL and Heald R (2010) Copyright © 2010. Image reproduced with permission of the Publisher, Elsevier B. V. Larger Image Printer Friendly View

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