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

Figure 7.

Figure 7

Mcl-1 contains a D-box motif that is required for its degradation during mitotic arrest. (A) Sequence alignment of putative Mcl-1 D-box motif compared with those from cyclin A, cyclin B1 and securin. The two residues in Mcl-1 mutated to alanine to yield the Mcl-1 D-box mutant (R207A/L210A) are indicated (arrows). (B) Sequence alignment of the C-terminal regions of Cdc20, Cdh1, APC10, Nek2A and Mcl-1. The I/M-R motif is underlined. (C) Mcl-1 D-box mutant is stable in cells arrested in mitosis. U2OS cells stably transfected with either wild-type Mcl-1 (WT), D-box or IR-deletion (ΔIR) mutants were treated for 2 h with nocodazole. Mitotic cells were then isolated and re-plated for a further 2 or 4 h so that cells were arrested in mitosis for a total of 2, 4 or 6 h. (D) Mcl-1 D-box and IR-deletion (ΔIR) mutants are degraded like wild-type Mcl-1 in interphase. Time course after addition of 40 μg/ml cycloheximide (CHX) to asynchronous cell cultures. (E) Mcl-1 D-box and IR-deletion (ΔIR) mutants are phosphorylated at Thr92 like wild-type Mcl-1 during mitotic arrest. U2OS cells stably transfected with either wild-type Mcl-1 (WT), D-box or IR-deletion (ΔIR) mutants were treated for 2 h with nocodazole before collection of mitotic cells (Noc) or left untreated (As). Flag-Mcl-1 was precipitated from cell lysates using Flag-agarose and immunoblotted for Mcl-1 phosphorylated at Thr92 or total Mcl-1. Cell lysates (Input) used for the precipitations are shown in the right panels.