Figure 8. Co-expression of either RANBP2... | Rockefeller University Press

Figure 8.

$Figure 8. Co-expression of either RANBP2 or CRM1 reduces importin-β–dependent mitotic abnormalities. (A) WB analysis of cell extracts transfected either with importin-β 1–876 (left lane in each panel) or with the indicated constructs (right lanes). Extracts were fractionated through 6% SDS-PAGE and constructs were detected using GFP antibody. (B) Mitotic cells transfected with importin-β 1–876 alone (top), or together with BPN, BPM, or CRM1. A trace of H2B-GFP was added to visualize chromosomes. Arrows point to chromosomes that fail to align. Z-stack (0.6–0.8 µm) images were flattened using the Maximum Intensity Projection tool (NIS-Elements 3.1; Nikon). Bar, 10 µm. (C) Frequency of mitotic defects in fixed cells transfected with importin-β alone or with RANBP2 fragments, or with CRM1 (740–2,000 counted cells per group, at least 5 experiments). **, highly significant (P < 0.01, χ2 test) differences between importin-β and vector (in red), and between cotransfected samples compared with importin-β only (in black). No statistical difference was observed between importin-β alone and cotransfection with BPN. (D) Co-expression of either BPM or CRM1, but not BPN, rescues RANGAP1 localization to KTs. Histograms represent the frequency of metaphases with delocalized RANGAP1 from KTs (at least 350 counted cells per group, 4 experiments). **, highly significant (P < 0.001, χ2 test) differences between importin-β and vector are indicated by red asterisks, and between cotransfected samples and importin-β alone by black asterisks. (E) Co-expression of either BPM or CRM1 reduces importin-β–dependent prometaphase delay and dynamic defects in video-recorded cells.$

Co-expression of either RANBP2 or CRM1 reduces importin-β–dependent mitotic abnormalities. (A) WB analysis of cell extracts transfected either with importin-β 1–876 (left lane in each panel) or with the indicated constructs (right lanes). Extracts were fractionated through 6% SDS-PAGE and constructs were detected using GFP antibody. (B) Mitotic cells transfected with importin-β 1–876 alone (top), or together with BPN, BPM, or CRM1. A trace of H2B-GFP was added to visualize chromosomes. Arrows point to chromosomes that fail to align. Z-stack (0.6–0.8 µm) images were flattened using the Maximum Intensity Projection tool (NIS-Elements 3.1; Nikon). Bar, 10 µm. (C) Frequency of mitotic defects in fixed cells transfected with importin-β alone or with RANBP2 fragments, or with CRM1 (740–2,000 counted cells per group, at least 5 experiments). **, highly significant (P < 0.01, χ² test) differences between importin-β and vector (in red), and between cotransfected samples compared with importin-β only (in black). No statistical difference was observed between importin-β alone and cotransfection with BPN. (D) Co-expression of either BPM or CRM1, but not BPN, rescues RANGAP1 localization to KTs. Histograms represent the frequency of metaphases with delocalized RANGAP1 from KTs (at least 350 counted cells per group, 4 experiments). **, highly significant (P < 0.001, χ² test) differences between importin-β and vector are indicated by red asterisks, and between cotransfected samples and importin-β alone by black asterisks. (E) Co-expression of either BPM or CRM1 reduces importin-β–dependent prometaphase delay and dynamic defects in video-recorded cells.