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	Figure 2. Inhibition of nuclear growth by human LAP2β fragment 1–408. (a) Nuclei assembled for 1.5 h in reactions containing the indicated concentration of fragment 1–408. The small phase-dense black dots (center of control nucleus) are coiled bodies, which form only in transport-competent nuclei. Upper panels show phase-contrast images of representative nuclei; lower panels show corresponding DNA, as visualized by Hoechst staining. (b) LAP2-arrested nuclei import a fluorescent karyophilic substrate. Nuclei were assembled for 2 h in the presence or absence of 3 μM LAP2 fragment 1–408, supplemented with rhodamine-conjugated nucleoplasmin, and viewed by epifluorescence 30 min later. As a negative control, WGA, which inhibits nuclear import, was added to samples 5 min before adding nucleoplasmin (right panels). Upper panels show phase-contrast images of representative nuclei; lower panels show the corresponding nuclei by rhodamine fluorescence, indicative of transport activity.
	Figure 3. Inhibition of nuclear assembly by human LAP2 fragment 1–187. (a) Nuclei assembled for 3 h in reactions containing the indicated concentration of fragment 1–187. Upper panels show phase-contrast images; lower panels show DNA as visualized by Hoechst, except the right-most panel which shows a second phase-contrast image of a nucleus inhibited by 10 μM fragment 1–187. (b) Nuclei were assembled in a reaction containing 30 μM fragment 1–187 for the indicated times. Membrane association with chromatin is delayed, but after 3 h the nuclei appear similar to those arrested by 5–10 μM fragment 1–187.
	Figure 4. TEM of nuclei inhibited by human LAP2 fragments. (a) Positive (buffer) control: nucleus assembled for 3 h. (b) Nucleus assembled for 3 h in a reaction containing 3 μM fragment 1–408. Insets in a and b are higher magnifications to show NPCs. Arrows in b indicate regions where the inner membrane appears to invaginate. (c and d) Nuclei assembled for 3 h in a reaction containing 10 μM LAP2 fragment 1–187. Arrowheads indicate NPCs. Bars, 500 nm.
	Figure 5. Lamin accumulation in nuclei inhibited by LAP2 fragments. (a) Western blot of nuclei assembled in the presence of the indicated LAP2 fragment. Nuclei were assembled for 3 h and pelleted; the pelleted proteins were separated by 12% SDS-PAGE and immunoblotted with a monoclonal anti-lamin antibody (see Materials and Methods). (b) Lamins detected by indirect immunofluorescence in nuclei assembled for 3 h in (left to right): a control reaction (buffer added), a reaction containing 3 μM LAP2 fragment 1–408, and a reaction containing 5 μM LAP2 fragment 1–187. Nuclei were processed for immunofluorescence as described in Materials and Methods. Upper panels, contrast images; middle panels, DNA visualized with Hoechst; bottom panels, lamins visualized by a Texas red–conjugated secondary antibody.
	Figure 6. DNA replication and lamin blots of nuclei inhibited by LAP2 fragments. Nuclei were assembled for 3 h in the presence of α[32P]dCTP plus either buffer, 3 μM LAP2β fragment 1–408, 5 μM LAP2 fragment 1–187, or 1 mM GTPγS. (a) Total DNA was prepared from aliquots of each sample, electrophoresed on a 0.8% agarose gel, and the gel dried and exposed to film (see Materials and Methods). Upper panel shows the autoradiograph of dried agarose gel; the lower panel shows the ethidium stain of the same gel as a control for DNA loading. (b) Protein from each sample was blotted and probed using a monoclonal anti-lamin antibody (see Materials and Methods). (c) Corresponding phase-contrast images of a typical nucleus from each sample in a.
	Figure 7. Replication of LAP2-arrested nuclei is aphidicolin-sensitive. Nuclei were assembled for 3 h with or without either LAP2 fragment 1–408 (3.3 μM) or aphidicolin (50 μM), in the presence of α[32P]dCTP. Reactions were digested with proteinase K, run on agarose gels, and label incorporated into high molecular weight DNA was quantitated by PhosphorImager (see Materials and Methods).
	Figure 8. BrdU density substitution experiment, and replication time course. (a) Density substitution experiment. Nuclei were assembled for 2 h in reactions containing BrdU and α[32P]dCTP, in the presence or absence of 3.3 μM fragment 1–408. The substituted DNA was purified and separated by centrifugation on equilibrium CsCl gradients (see Materials and Methods). Replicated DNA (cpm of incorporated α[32P]dCTP) in each gradient fraction is plotted against gradient fractions, which had equal densities as determined by refractive index measurements. HL indicates the density of heavy–light DNA (1.746 g/ml), and HH the expected density of heavy–heavy DNA (1.80 g/ml; Walter et al., 1998). Filled circles indicate reactions lacking LAP2; open circles indicate reactions containing 3.3 μM LAP2 fragment 1–408. (b) Replication time course. Nuclei were assembled in reactions containing α[32P]dCTP, in the presence or absence of fragment 1–408. Aliquots were removed every 15 min for 3 h, and processed by running purified DNA on agarose gel, and quantitating the incorporated [32P]dCTP by PhosphorImager (see Materials and Methods). Filled circles indicate reactions lacking LAP2; open circles indicate reactions containing 3.3 μM LAP2 fragment 1–408.
	Figure 9. Comparison of predicted Xenopus LAP2 proteins to human LAP2β. (a) Schematic diagrams of translated Xenopus cDNAs and human LAP2β protein. Homologous regions between Xenopus and human LAP2 proteins are shaded similarly. Numbers denote residues that begin and end each block of homology. The black box in the striped COOH-terminal region denotes the predicted transmembrane span. The predicted masses of the proteins encoded by the Xenopus LAP2 cDNAs are 62.841 kD (clone 2), 46.363 (clone 3), and 58.674 kD (clone 4). DNA sequence data are available from GenBank under accession numbers AF048815 (clone 2), AF048816 (clone 3), and AF048817 (clone 4). (b) Alignment of human LAP2β and Xenopus LAP2-clone 2 proteins using BOXSHADE (see Materials and Methods). Black shading indicates identity, gray shading indicates similarity.
	Figure 10. Inhibition of nuclear assembly by Xenopus LAP2 fragment 1–164. Nuclei were assembled for 3 h in reactions containing the indicated concentration of fragment 1–164. Upper panels show phase-contrast images; lower panels show DNA as visualized by Hoechst. Xenopus fragment 1–164 gives the same arrest phenotype as human fragment 1–187, but at lower concentrations.

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