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.