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1.
Figure 2

Figure 2. From: Yeast Nucleoporins Involved in Passive Nuclear Envelope Permeability.

Steady-state localization of cNLS-GFP in wt, nup170-Δ, and nup188-Δ cells was determined before induction of GAL1-SSA1 (23°C), after induction (23°C +SSA1), and incubated on ice after induction (0°C +SSA1). GFP fluorescence and Hoechst stain images were obtained by confocal microscopy (see Materials and Methods).

Nataliya Shulga, et al. J Cell Biol. 2000 May 29;149(5):1027-1038.
2.
Figure 6

Figure 6. From: Yeast Nucleoporins Involved in Passive Nuclear Envelope Permeability.

Effects of chilling on the nuclear localization of pNLS-GFP in wt, nup188-Δ, and nup170-Δ cells. Cells were grown at 23°C and incubated at 0°C as described in Materials and Methods. GFP fluorescence and Hoechst stain images were obtained by confocal microscopy.

Nataliya Shulga, et al. J Cell Biol. 2000 May 29;149(5):1027-1038.
3.
Figure 1

Figure 1. From: Yeast Nucleoporins Involved in Passive Nuclear Envelope Permeability.

Export kinetics of cNLS-GFP as a function of temperature in azide/deoxyglucose. Cells expressing cNLS-GFP were grown at 24°C, pelleted, washed, and suspended in glucose-free SC medium containing 10 mM azide and 10 mM 2-deoxyglucose that had been warmed or chilled at the assay temperature. Export was quantified as described in Materials and Methods.

Nataliya Shulga, et al. J Cell Biol. 2000 May 29;149(5):1027-1038.
4.
Figure 5

Figure 5. From: Yeast Nucleoporins Involved in Passive Nuclear Envelope Permeability.

Effects of chilling, GAL1-SSA1 expression, and azide/deoxyglucose on the nuclear localization of rgNLS-GFP in wt, nup188-Δ, and nup170-Δ cells. A, Localization of rgNLS-GFP in wt, nup170-Δ, and nup188-Δ cells was determined before induction of GAL1-SSA1 (23°C), after induction (23°C +SSA1), after incubation on ice after induction (0°C +SSA1), and after incubation in azide/deoxyglucose. GFP and DIC images were captured by confocal microscopy (see Materials and Methods). B, Kinetic import assay of rgNLS-GFP in wt and mutant cells after equilibration in azide/deoxyglucose. Import was assayed in nup170-Δ and nup188-Δ cells ± GAL1-SSA1 induction. Kinetic assays were performed at 37°C as described in Materials and Methods.

Nataliya Shulga, et al. J Cell Biol. 2000 May 29;149(5):1027-1038.
5.
Figure 5

Figure 5. From: Yeast Nucleoporins Involved in Passive Nuclear Envelope Permeability.

Effects of chilling, GAL1-SSA1 expression, and azide/deoxyglucose on the nuclear localization of rgNLS-GFP in wt, nup188-Δ, and nup170-Δ cells. A, Localization of rgNLS-GFP in wt, nup170-Δ, and nup188-Δ cells was determined before induction of GAL1-SSA1 (23°C), after induction (23°C +SSA1), after incubation on ice after induction (0°C +SSA1), and after incubation in azide/deoxyglucose. GFP and DIC images were captured by confocal microscopy (see Materials and Methods). B, Kinetic import assay of rgNLS-GFP in wt and mutant cells after equilibration in azide/deoxyglucose. Import was assayed in nup170-Δ and nup188-Δ cells ± GAL1-SSA1 induction. Kinetic assays were performed at 37°C as described in Materials and Methods.

Nataliya Shulga, et al. J Cell Biol. 2000 May 29;149(5):1027-1038.
6.
Figure 4

Figure 4. From: Yeast Nucleoporins Involved in Passive Nuclear Envelope Permeability.

Import kinetics of cNLS-GFP at 0°C. Wt, nup170-Δ, and nup188-Δ cells were treated for 40 min with 10 mM 2-deoxyglucose at 30°C to equilibrate cNLS-GFP. Cells were then washed and resuspended in ice-cold complete medium and incubated at 0°C. Cells were harvested at various times and assayed for cNLS-GFP nuclear accumulation as described in Materials and Methods. Also shown are fluorescence (GFP) and light (DIC) images of wt cells before and after 2-deoxyglucose-induced equilibration, and after 20 h on ice.

Nataliya Shulga, et al. J Cell Biol. 2000 May 29;149(5):1027-1038.
7.
Figure 4

Figure 4. From: Yeast Nucleoporins Involved in Passive Nuclear Envelope Permeability.

Import kinetics of cNLS-GFP at 0°C. Wt, nup170-Δ, and nup188-Δ cells were treated for 40 min with 10 mM 2-deoxyglucose at 30°C to equilibrate cNLS-GFP. Cells were then washed and resuspended in ice-cold complete medium and incubated at 0°C. Cells were harvested at various times and assayed for cNLS-GFP nuclear accumulation as described in Materials and Methods. Also shown are fluorescence (GFP) and light (DIC) images of wt cells before and after 2-deoxyglucose-induced equilibration, and after 20 h on ice.

Nataliya Shulga, et al. J Cell Biol. 2000 May 29;149(5):1027-1038.
8.
Figure 3

Figure 3. From: Yeast Nucleoporins Involved in Passive Nuclear Envelope Permeability.

Temperature dependence of cNLS-GFP nuclear accumulation in wt and mutant cells. A, Dynamics of cNLS-GFP localization in wt and nup188-Δ cells ± GAL1-SSA1 induction were quantified as described in Materials and Methods. At time = 0, cells were shifted from 23°C to an ice bath (0°C). Wt cells were maintained at 0°C for the duration of the time course. Arrows on the time line indicate when nup188-Δ cells were shifted from 0 to 23°C (15 min) and then back to 0°C (30 min). B, Relaxation kinetics of cNLS-GFP redistribution in nup188-Δ + SSA1 cells after shifting initial incubation temperature from 30 to 20, 10, or 0°C. C, Export kinetics of cNLS-GFP at 0°C in additional null strains.

Nataliya Shulga, et al. J Cell Biol. 2000 May 29;149(5):1027-1038.
9.
Figure 3

Figure 3. From: Yeast Nucleoporins Involved in Passive Nuclear Envelope Permeability.

Temperature dependence of cNLS-GFP nuclear accumulation in wt and mutant cells. A, Dynamics of cNLS-GFP localization in wt and nup188-Δ cells ± GAL1-SSA1 induction were quantified as described in Materials and Methods. At time = 0, cells were shifted from 23°C to an ice bath (0°C). Wt cells were maintained at 0°C for the duration of the time course. Arrows on the time line indicate when nup188-Δ cells were shifted from 0 to 23°C (15 min) and then back to 0°C (30 min). B, Relaxation kinetics of cNLS-GFP redistribution in nup188-Δ + SSA1 cells after shifting initial incubation temperature from 30 to 20, 10, or 0°C. C, Export kinetics of cNLS-GFP at 0°C in additional null strains.

Nataliya Shulga, et al. J Cell Biol. 2000 May 29;149(5):1027-1038.
10.
Figure 7

Figure 7. From: Yeast Nucleoporins Involved in Passive Nuclear Envelope Permeability.

NE sieving limits in wt, nup188-Δ, and nup170-Δ cells with NES-GFP molecular size probes. A, Localization of NES-GFP66 at 23 and 0°C in wt, nup188-Δ, and nup170-Δ cells. NES-GFP66 fluorescence (GFP) and Hoechst stain images were obtained by confocal microscopy. B, Time course of reexport of equilibrated NES-GFP66 in nup188-Δ cells. nup188-Δ cells incubated at 0°C for 1 h were transferred to a slide and the localization of NES-GFP66 observed as the cells warmed to room temperature (∼23°C). C, Quantification of nucleocytoplasmic distributions of NES-GFP size probes in wt, nup188-Δ, and nup170-Δ cells at 23 and 0°C. NES-GFP66, 81-, and 126-kD probes were expressed in wt and mutant cells at 23°C and then shifted to 0°C for 1 h. The top value in each ratio is the [C]/[N] ratio at 23°C and the bottom value is the [C]/[N] ratio at 0°C (the values of the ratios of these ratios are indicated to the right). In each case, nuclear and cytoplasmic levels of GFP fluorescence ([C]/[N]) at 23 and 0°C were quantified using the averaged pixel density of three different areas within the nuclei and cytoplasms of 10–15 different cells using the region tool in MetaMorph.

Nataliya Shulga, et al. J Cell Biol. 2000 May 29;149(5):1027-1038.

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