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Figure 3. The Bub1–Mad1 interaction is crucial for checkpoint activation in human cells.(A) HeLa cells expressing Myc-Bub1 transgenes were treated with nocodazole and MG132 in the presence or absence of reversine. Myc-Bub1 proteins were immunoprecipitated and blotted with the indicated antibodies. (B) Mitotic indices of cells expressing Bub1 transgenes that were transfected with the indicated Bub1 siRNA and treated with nocodazole in the presence or absence of the Plk1 inhibitor BI2536. Error bars, s.d. (n = 3 independent experiments). ***p<0.001; ****p<0.0001; Student’s t-test. (C) Lysates of cells in (B) were blotted with the indicated antibodies. Endo., endogenous. (D) Mitotic indices of cells expressing the indicated Mis12–Mad1 fusion proteins that were treated with reversine or siRNAs against BubR1 or Mad2. MIM/5A, Mad1 mutant with its MIM (541KVLHM545) changed to five alanines. Error bars, s.d. (n = 3 independent experiments). ***p<0.001; ****p<0.0001; Student’s t-test. (E) Lysates of cells in (D) were blotted with the indicated antibodies.DOI: http://dx.doi.org/10.7554/eLife.22513.006 Image published in: Ji Z et al. (2017) © 2017, Ji et al. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View

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