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. 2003 Feb 3;160(3):341–353. doi: 10.1083/jcb.200211048

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Copyright © 2003, The Rockefeller University Press

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Figure 3. — The mitotic checkpoint requires a full complement of Rae1. (A) Analysis of Rae1^+/+ and Rae1^+/− MEF lines for Rae1, Bub3, and Mad2 protein levels by Western blotting (4–20% polyacrylamide gel). 100 μg of total protein extract from each MEF cell line was used in the analysis. For probes, we used a rabbit antibody against mouse Rae1(188–368), a rabbit antibody against mouse Bub3(145–276), and a mouse monoclonal antibody against human Mad2 that recognizes mouse Mad2 (BD Biosciences). (B) Growth curves of primary MEFs. Data shown are means and standard deviations derived from three Rae1^+/+ and three Rae1^+/− MEF lines. (C) Mitotic index of nocodazole-treated Rae1^+/− and Rae1^+/+ MEF cell lines (n = 3 for each genotype). (D) Representative phase contrast images of Rae1^+/+ and Rae1^+/− MEF cultures before and after 4 h of nocodazole exposure. (E) Representative FACS^® profiles of propidium-stained unsynchronized Rae1^+/+ (panels 1–3) and Rae1^+/− MEFs (panels 4–6). Durations of nocodazole treatment (80 ng/ml) and recovery intervals are indicated. (F) Cyclin B–associated Cdc2 kinase activity of synchronized MEF cells at indicated time points after release into nocodazole.