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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 4. — Affinity-purified GST–hBUBR1:MCC inhibits APC/C. (A) GST–hBUBR1 is associated with the MCC. A GST–hBUBR1 construct was transfected into cells and the protein was affinity purified using glutathione beads. Affinity-purified GST–hBUBR1 (lane 3) and the remaining supernatant (lane 4) were probed for hBUBR1, hBUB3, CDC20, and MAD2. In parallel, a GST construct was transfected and the affinity-purified GST (lane 1) and the remaining supernatant (lane 2) was probed with the same antibodies and a GST antibody to confirm expression of GST in transfected cells. The black arrowhead points to endogenous hBUBR1 and the open arrowheads point to GST–hBUBR1 and its degradation product (as confirmed by GST Western blot, unpublished data). (B) Affinity-purified GST–hBUBR1:MCC inhibits APC/C. Mitotic APC/C (lane 2) was incubated with GST (lane 3), conventionally purified MCC (lane 4), and GST-MCC (lane 5) and assayed for ubiquitin ligase activity. A control reaction lacking APC/C (lane 1) provided the level of input substrate that was used for calculating APC/C activity in the various reactions.

Figure 4. — Affinity-purified GST–hBUBR1:MCC inhibits APC/C. (A) GST–hBUBR1 is associated with the MCC. A GST–hBUBR1 construct was transfected into cells and the protein was affinity purified using glutathione beads. Affinity-purified GST–hBUBR1 (lane 3) and the remaining supernatant (lane 4) were probed for hBUBR1, hBUB3, CDC20, and MAD2. In parallel, a GST construct was transfected and the affinity-purified GST (lane 1) and the remaining supernatant (lane 2) was probed with the same antibodies and a GST antibody to confirm expression of GST in transfected cells. The black arrowhead points to endogenous hBUBR1 and the open arrowheads point to GST–hBUBR1 and its degradation product (as confirmed by GST Western blot, unpublished data). (B) Affinity-purified GST–hBUBR1:MCC inhibits APC/C. Mitotic APC/C (lane 2) was incubated with GST (lane 3), conventionally purified MCC (lane 4), and GST-MCC (lane 5) and assayed for ubiquitin ligase activity. A control reaction lacking APC/C (lane 1) provided the level of input substrate that was used for calculating APC/C activity in the various reactions.