Figure 4. NUP88 sequesters NUP98-RAE1 away from APC/C^CDH1 prior to mitotic entry when overexpressed.

(A) Coprecipitation analysis of RO-3306–treated HeLa cells immunoprecipitated with an anti-NUP98 Ab. (B) Coprecipitation analysis of RO-3306–treated HeLa cells transduced with pTRIPZ–HA-Nup88 (NUP88) or pTRIPZ empty vector (EV) lentiviruses and immunoprecipitated with anti–HA affinity matrix. (C) Coprecipitation analysis of cells in B immunoprecipitated with an anti-NUP98 Ab. Values represent NUP88/EV protein quantification ratios. (D) Images of WT MEFs immunostained for APC6, CDC27, or CDH1. p-HH3 (Ser10) was used to identify cells in the G₂ phase. (E) Western blot analysis of cytoplasmic (C) and nuclear (N) fractions from MEFs of the indicated genotypes. (F) Same as in E. (G) Coprecipitation analysis of cytoplasmic fractions from MEFs of the indicated genotypes immunoprecipitated with anti–HA affinity matrix. (H) Same as in G, except immunoprecipitated with an anti-NUP88 Ab. (I) Live-cell–imaging analysis of chromosome segregation errors in H2B-mRFP–positive MEFs of the indicated genotypes. (J) Mitotic checkpoint analysis of MEFs in I challenged with nocodazole. (K) Western blot of RO-3306–treated or (L) nocodazole-arrested MEFs of the indicated genotypes. Analysis in I was performed on 3 independent lines per genotype (25 cells/line). Analysis in J was performed on 3 independent lines per genotype (>10 cells/line). Data represent the mean ± SEM. Western blots are representative of 3 independent experiments. Cyclin B1, cyclin A2, and actin blots in K and L are identical to those in Figure 5, G and H. WT and NUP98 RAE1 values in I and J are identical to those in Figure 2, F and H. Statistical significance in I and J was determined using a 2-tailed, unpaired t test. *P < 0.05, **P < 0.01, and ***P < 0.001. Scale bar: 10 μm. WCE, whole-cell extract.