Figure 8. Brr6 and Brl1 accumulate at NPC assembly intermediates. (A) Protein level of yeGFP-Brl1 of the indicated cells represented in B. Tub2 is loading control. (B) Images of cells incubated at 37°C for 3 h. The enlargements (bottom) show NEs that were used for the plot profiles (right). Arrowheads indicate colocalization of yeGFP-Brl1 and Nup85-tdTomato. Bars: (overview) 5 µm; (enlargement) 1 µm. (C) Quantification of cells from B. Error bars: SD (n > 230); three independent experiments. (D and E) Immuno-EM of cells incubated for 3 h at 37°C. Localization of Brl1, Brr6 (10 nm gold, anti-Brl1, and anti-GFP), and Nsp1 (15 nm, anti-Nsp1) at NPC intermediates and herniations. Cartoons illustrate the morphology of the NE evaginations with gold labeling. N, nucleus; C, cytoplasm. Bars, 100 nm. (F) Deepness of NPC intermediates and herniations of Nsp1-labeled cells was measured as indicated by the bidirectional arrows in the cartoon. ONM–INM distance was measured from BRR6 BRL1 WT cells near NPCs. ****, P ≤ 0.0001. (G) Diameter of NPC intermediates and herniations (see cartoon) was quantified from td-brr6, td-brl1, and td-brr6 td-brl1 cells. The diameter of normal NPCs was measured from BRR6 BRL1 WT cells. *, P ≤ 0.05; ***, P ≤ 0.001; ****, P ≤ 0.0001; ns, not significant.
Figure 8.

Brr6 and Brl1 accumulate at NPC assembly intermediates. (A) Protein level of yeGFP-Brl1 of the indicated cells represented in B. Tub2 is loading control. (B) Images of cells incubated at 37°C for 3 h. The enlargements (bottom) show NEs that were used for the plot profiles (right). Arrowheads indicate colocalization of yeGFP-Brl1 and Nup85-tdTomato. Bars: (overview) 5 µm; (enlargement) 1 µm. (C) Quantification of cells from B. Error bars: SD (n > 230); three independent experiments. (D and E) Immuno-EM of cells incubated for 3 h at 37°C. Localization of Brl1, Brr6 (10 nm gold, anti-Brl1, and anti-GFP), and Nsp1 (15 nm, anti-Nsp1) at NPC intermediates and herniations. Cartoons illustrate the morphology of the NE evaginations with gold labeling. N, nucleus; C, cytoplasm. Bars, 100 nm. (F) Deepness of NPC intermediates and herniations of Nsp1-labeled cells was measured as indicated by the bidirectional arrows in the cartoon. ONM–INM distance was measured from BRR6 BRL1 WT cells near NPCs. ****, P ≤ 0.0001. (G) Diameter of NPC intermediates and herniations (see cartoon) was quantified from td-brr6, td-brl1, and td-brr6 td-brl1 cells. The diameter of normal NPCs was measured from BRR6 BRL1 WT cells. *, P ≤ 0.05; ***, P ≤ 0.001; ****, P ≤ 0.0001; ns, not significant.

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