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. 2001 Sep 3;154(5):925–936. doi: 10.1083/jcb.200102093

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Copyright © 2001, The Rockefeller University Press

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Figure 5. — Characterization of the MCC. (A) Estimation of MAD2 content in MCC. Quantitative immunoblot of purified MCC with known amounts of recombinant MAD2. Recombinant (His) 6-tagged MAD2 migrated slower than native MAD2. (B) Comparison of APC/C inhibitory activity between the recombinant oligomeric MAD2 and purified MCC. Increasing amounts of oligomeric MAD2 and purified MCC were added to the APC/C assay to compare inhibitory activities. Phosphorimage of the APC/C reactions (upper panel) was quantified to determine the amount of recombinant MAD2 and MCC required to achieve equivalent levels of inhibition (lower panel). (C) MCC subunits exist in near equal stoichiometry. HeLa cells were labeled in ³⁵S-Trans label for 6 h and mitotic and interphase cells were separated and then incubated with nonimmune IgG or hBUBR1 antibodies. Phosphorimage of the immunoprecipitate obtained from mitotic cells shows the radiolabeled hBUBR1, hBUB3, MAD2, and CDC20. The counts from each subunit were normalized to their cysteine and methionine contents (without initiating Met) to estimate their stoichiometry.