Journal: The Journal of Cell Biology

Article Title: Lamin-binding Fragment of LAP2 Inhibits Increase in Nuclear Volume during the Cell Cycle and Progression into S Phase

doi:

Figure Lengend Snippet: Analysis of LAP2 fragments used for microinjection by SDS-PAGE. Samples of recombinant proteins used for microinjection were electrophoresed on a 10% SDS gel, that was stained with Coomassie blue. MW , molecular weight standards; lane 1 , MBP-LAP2 (298–373); lane 2 , MPB-LAP2 (1–88); lane 3 , His-tagged LAP2 (1–398).

Article Snippet: To obtain an expression plasmid encoding the maltose-binding protein (MBP) fused to residues 298–373 of LAP2, the cDNA sequence encoding the corresponding region of LAP2 was excised by BamHI and HindIII digestion from the vector pGEX-LAP2 (298–373), and then was inserted into the vector pMALc ( New England Biolabs, Inc. , Beverly, MA).

Techniques: SDS Page, Recombinant, SDS-Gel, Staining, Molecular Weight

Journal: The Journal of Cell Biology

Article Title: Lamin-binding Fragment of LAP2 Inhibits Increase in Nuclear Volume during the Cell Cycle and Progression into S Phase

doi:

Figure Lengend Snippet: Effect of microinjected LAP2 fragments on nuclear reformation and lamin assembly at the end of mitosis. Metaphase HeLa cells were injected with a variety of proteins and incubated for 2 h at 37°C before fixation and immunofluorescent staining. All injectates contained rabbit IgG to mark the injected cells. In addition, the injectates contained: A and E , LAP2 (1–398); B and F , rabbit IgG only; C and G , MBP-LAP2 (298–373); D and H , MBP-LAP2 (1–88). Shown are phase contrast images ( left column ), or epifluorescence images staining with anti–rabbit IgG ( middle column ), and with antilamins ( right column ; A–D , antilamins A/C; E–H , antilamin B). Bar, 20 μm.

Article Snippet: To obtain an expression plasmid encoding the maltose-binding protein (MBP) fused to residues 298–373 of LAP2, the cDNA sequence encoding the corresponding region of LAP2 was excised by BamHI and HindIII digestion from the vector pGEX-LAP2 (298–373), and then was inserted into the vector pMALc ( New England Biolabs, Inc. , Beverly, MA).

Techniques: Injection, Incubation, Staining

Journal: The Journal of Cell Biology

Article Title: Lamin-binding Fragment of LAP2 Inhibits Increase in Nuclear Volume during the Cell Cycle and Progression into S Phase

doi:

Figure Lengend Snippet: Effect of microinjected LAP2 fragments on reassembly of LAPs and pore complex proteins at the end of mitosis. Metaphase HeLa cells were injected with rabbit IgG plus MBP-LAP2 (1–88) ( A–C ) or MPB-LAP2 (298–373) ( D–F ). After incubation for 2 h at 37°C, cells were fixed and stained for immunofluorescence microscopy. Shown are confocal fluorescence images of staining with anti–rabbit IgG ( left image in A–F ) and with antibodies against various NE proteins ( right image in A–F ): A and D , anti-LAP1; B and E , anti-LAP2; C and F , RL1. Bar, 20 μm.

Article Snippet: To obtain an expression plasmid encoding the maltose-binding protein (MBP) fused to residues 298–373 of LAP2, the cDNA sequence encoding the corresponding region of LAP2 was excised by BamHI and HindIII digestion from the vector pGEX-LAP2 (298–373), and then was inserted into the vector pMALc ( New England Biolabs, Inc. , Beverly, MA).

Techniques: Injection, Incubation, Staining, Immunofluorescence, Microscopy, Fluorescence

Journal: The Journal of Cell Biology

Article Title: Lamin-binding Fragment of LAP2 Inhibits Increase in Nuclear Volume during the Cell Cycle and Progression into S Phase

doi:

Figure Lengend Snippet: Electron microscopy of G1 phase cells injected with LAP2 (298–373) at metaphase. Shown are thin section electron micrographs depicting the nucleus of cells that were injected with LAP2 (298–373) at metaphase and returned to culture for 2 h. Nuclear ( N ) and cytoplasmic ( C ) areas are indicated. A shows a tangential section of the nuclear surface, whereas B and C show cross-sections of the nucleus. Nuclear pore complexes are indicated by arrows. Bars, 1 μm.

Article Snippet: To obtain an expression plasmid encoding the maltose-binding protein (MBP) fused to residues 298–373 of LAP2, the cDNA sequence encoding the corresponding region of LAP2 was excised by BamHI and HindIII digestion from the vector pGEX-LAP2 (298–373), and then was inserted into the vector pMALc ( New England Biolabs, Inc. , Beverly, MA).

Techniques: Electron Microscopy, Injection

Journal: The Journal of Cell Biology

Article Title: Lamin-binding Fragment of LAP2 Inhibits Increase in Nuclear Volume during the Cell Cycle and Progression into S Phase

doi:

Figure Lengend Snippet: EM of G1 phase cells injected with LAP2 (1–88) at metaphase. Shown are thin section electron micrographs depicting the nucleus of cells injected with LAP2 (1–88) at metaphase and returned to culture for 2 h. Nuclear ( N ) and cytoplasmic ( C ) areas are indicated. A shows a tangential section of the nuclear surface, whereas B and C show cross-sections of the nucleus. Bars, 1 μm.

Article Snippet: To obtain an expression plasmid encoding the maltose-binding protein (MBP) fused to residues 298–373 of LAP2, the cDNA sequence encoding the corresponding region of LAP2 was excised by BamHI and HindIII digestion from the vector pGEX-LAP2 (298–373), and then was inserted into the vector pMALc ( New England Biolabs, Inc. , Beverly, MA).

Techniques: Injection

Journal: The Journal of Cell Biology

Article Title: Lamin-binding Fragment of LAP2 Inhibits Increase in Nuclear Volume during the Cell Cycle and Progression into S Phase

doi:

Figure Lengend Snippet: Analysis of nuclear protein import in interphase cells injected with LAP2 fragments. (A and B) Interphase HeLa cells in exponentially growing populations were injected with either MPB-LAP2 (298–373) ( A ) or MBP-LAP2 (1–88) ( B ) together with fluorescent rabbit IgG. After 2 h, the injected cells were given a second injection with FITC-NLS-BSA. The cells were fixed 30 min later and examined. ( C and D ) Mitotic HeLa cells were selected by mitotic shake off and were returned to culture. After 2 h, they were injected with either MPB-LAP2 (298–373) ( C ) or MBP-LAP2 (1–88) ( D ). 10 h later, the injected cells were given a second injection with FITC-NLS-BSA. The cells were fixed 30 min later and examined. Shown are phase contrast images ( left column ), epifluorescence images of FITC-NLS-BSA ( middle column ), and epifluorescence images of rhodamine-IgG ( right column ). Bars, 20 μm.

Article Snippet: To obtain an expression plasmid encoding the maltose-binding protein (MBP) fused to residues 298–373 of LAP2, the cDNA sequence encoding the corresponding region of LAP2 was excised by BamHI and HindIII digestion from the vector pGEX-LAP2 (298–373), and then was inserted into the vector pMALc ( New England Biolabs, Inc. , Beverly, MA).

Techniques: Injection

Journal: The Journal of Cell Biology

Article Title: Lamin-binding Fragment of LAP2 Inhibits Increase in Nuclear Volume during the Cell Cycle and Progression into S Phase

doi:

Figure Lengend Snippet: Effect of injected LAP2 fragments cells on interphase nuclear growth and progression to S phase. Mitotic HeLa cells were obtained by shake off, and returned to culture. Some cells were injected with MBP-LAP2 (298–373) either 2 h ( A and E ) or 10 h ( B ) later. Other cells were injected with MBP-LAP2 (1–88) either 2 h ( C ) or 10 h ( D ) later. All injectates also contained rabbit IgG. 12 h after the mitotic cells were returned to culture, the cultures in A–D were pulse labeled with BrdU for 30 min, and were fixed and processed for immunofluorescence microscopy as indicated. The culture in E was fixed at 12 h. Shown are phase contrast images ( A–D , left column ), or epifluorescence images of staining with anti-BrdU ( A–D , middle column ), and with anti– rabbit IgG ( A–D , right column ). E shows confocal fluorescence images of staining with antilamins A/C ( left ) and with anti-IgG ( right ). Bars, 10 μm.

Article Snippet: To obtain an expression plasmid encoding the maltose-binding protein (MBP) fused to residues 298–373 of LAP2, the cDNA sequence encoding the corresponding region of LAP2 was excised by BamHI and HindIII digestion from the vector pGEX-LAP2 (298–373), and then was inserted into the vector pMALc ( New England Biolabs, Inc. , Beverly, MA).

Techniques: Injection, Labeling, Immunofluorescence, Microscopy, Staining, Fluorescence

Journal: The Journal of Cell Biology

Article Title: Lamin-binding Fragment of LAP2 Inhibits Increase in Nuclear Volume during the Cell Cycle and Progression into S Phase

doi:

Figure Lengend Snippet: Effect of LAP2 (298–373) on Nuclear Volume Increase during the Cell Cycle

Article Snippet: To obtain an expression plasmid encoding the maltose-binding protein (MBP) fused to residues 298–373 of LAP2, the cDNA sequence encoding the corresponding region of LAP2 was excised by BamHI and HindIII digestion from the vector pGEX-LAP2 (298–373), and then was inserted into the vector pMALc ( New England Biolabs, Inc. , Beverly, MA).

Techniques: Injection, Sampling

Journal: The Journal of Cell Biology

Article Title: Lamin-binding Fragment of LAP2 Inhibits Increase in Nuclear Volume during the Cell Cycle and Progression into S Phase

doi:

Figure Lengend Snippet: Quantitative analysis of effect of injected LAP2 (298– 373) on progression of cells into S phase. ( A ) Kinetics of S phase progression. After selection of mitotic HeLa cells by shake off, the cells were returned to culture in medium containing BrdU and were fixed at various times and processed for immunofluorescence microscopy with anti-BrdU. The percentage of cells showing BrdU staining is indicated. ( B ) Mitotic cells obtained by shake off were returned to culture, and cells were injected with LAP2 (298-373) at 2 h ( black bar ), or at 10 h ( white bar ), or with LAP2 (1–88) at 2 h ( lightly shaded bar ). The cultures injected with LAP2 (298–373) and LAP2 (1–88) at 2 h, and a control uninjected culture ( darkly shaded bar ), were grown in medium containing BrdU from 2–12 h, and the cells were then fixed and processed for immunofluorescence microscopy with anti-BrdU. The culture injected with LAP2 (298–373) at 10 h was pulse labeled with BrdU for 30 min at 12 hr, and the cells were then fixed and stained with anti-BrdU. Shown are the percent of each population that incorporated BrdU.

Article Snippet: To obtain an expression plasmid encoding the maltose-binding protein (MBP) fused to residues 298–373 of LAP2, the cDNA sequence encoding the corresponding region of LAP2 was excised by BamHI and HindIII digestion from the vector pGEX-LAP2 (298–373), and then was inserted into the vector pMALc ( New England Biolabs, Inc. , Beverly, MA).

Techniques: Injection, Selection, Immunofluorescence, Microscopy, BrdU Staining, Labeling, Staining