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. 2010 Jun 4;29(14):2407–2420. doi: 10.1038/emboj.2010.112

Figure 6.

Figure 6

Destruction of Mcl-1 during mitotic arrest is mediated by Cdc20. (A) Cdc20 is required for Mcl-1 destruction in cells arrested in mitosis. Cdc20 was depleted from U2OS cells using siRNA, then cells were synchronised in a mitotic arrest by treatment with nocodazole for 2 h followed by collection of rounded-up mitotic cells and further treatment with nocodazole for a total of 4 or 6 h. Cells treated with a luciferase siRNA (Luc) were used as a control. (B) APC3 is required for Mcl-1 destruction in cells arrested in mitosis. APC3 was depleted from U2OS cells using siRNA, then cells were synchronised as in (A). Luciferase siRNA (Luc) was used as a control. (C) Depletion of Cdh1 does not prevent Mcl-1 degradation during mitotic arrest. Cdh1 was depleted from U2OS cells using siRNA, then cells were synchronised in mitotic arrest by treatment with nocodazole for 2 h followed by collection of rounded-up mitotic cells and further treatment with nocodazole for a total of 6 h. (D) Cdc20 interacts with Mcl-1 in interphase and mitosis. U2-Mcl-1-WT cells were synchronised by a single thymidine block followed by release for 9 h before addition of nocodazole (100 ng/ml) and MG132 (10 μM) for 3 h. Flag-Mcl-1 was precipitated from mitotic (NM) and adherent (NA) cells using Flag-agarose and immunoblotted for Cdc20 and Mcl-1; HA-agarose was used as a control (Ctrl) for the precipitation (right panels). Cell lysates (Input) used for the precipitations are shown in the left panels.