Figure 3. The ΔKILR motif interacts with the APC/C in a different manner compared with the IR tail and the C box. (A and B) The IR tail and C box motif in Cdc20 are required to interact with the APC/C. (A) HeLa cells expressing 3×Flag–wild-type, ΔIR, or C box mutant Cdc20 from an inducible promoter were treated with siRNA against Cdc20 and synchronized at mitosis as in Fig. 2 B. The APC/C was immunoprecipitated with anti-APC4 antibodies and analyzed by quantitative immunoblotting. Results are representative of three independent experiments. (B) In vitro translated (IVT) full-length 3×Flag–wild type, ΔIR, or C box mutants of Cdc20 were tested for their ability to bind to the APC/C as in Fig. 2 C. Results are representative of four independent experiments. (C and D) The ΔIR and C box mutants of Cdc20 can still interact with the APC/C when part of the MCC. (C) HeLa cell line expressing inducible 3×Flag-tagged wild-type, ΔIR, C box, or ΔKILR mutant Cdc20 were treated with siRNA against Cdc20, arrested at prometaphase with 0.33 µM nocodazole, and harvested by mitotic shake off. The APC/C was immunoprecipitated using anti-APC4 antibodies and analyzed by quantitative immunoblotting. Results are representative of three independent experiments. (D) HeLa cell lines expressing inducible 3×Flag-tagged wild-type or ΔIR mutant Cdc20 were treated with siRNA against Cdc20 or Cdc20 and APC3, arrested at prometaphase, and analyzed as in C. (E and F) The ΔKILR mutant is not able to form the MCC or bind to the APC/C. (E) HeLa cell lines expressing inducible 3×Flag-Cdc20wt or Cdc20ΔKILR were treated with siRNA against Cdc20, arrested in prometaphase with 0.33 µM nocodazole, and harvested by mitotic shake off. Extracts were analyzed by size-exclusion chromatography on a Sepharose 6 column, and fractions were analyzed by quantitative immunoblotting with antibodies against Cdc20. The peaks of APC/C and MCC migration are indicated. Results are representative of two independent experiments. (F) Distributions of wild-type Cdc20 and ΔKILR mutant with the sum of Cdc20 intensities set to 1. Immunoblotting with antibodies against APC3, Cdc20, BubR1, and Mad2 is shown in Fig. S3. end, endogenous; IP, immunoprecipitation; WT, wild type.
Figure 3.

The ΔKILR motif interacts with the APC/C in a different manner compared with the IR tail and the C box. (A and B) The IR tail and C box motif in Cdc20 are required to interact with the APC/C. (A) HeLa cells expressing 3×Flag–wild-type, ΔIR, or C box mutant Cdc20 from an inducible promoter were treated with siRNA against Cdc20 and synchronized at mitosis as in Fig. 2 B. The APC/C was immunoprecipitated with anti-APC4 antibodies and analyzed by quantitative immunoblotting. Results are representative of three independent experiments. (B) In vitro translated (IVT) full-length 3×Flag–wild type, ΔIR, or C box mutants of Cdc20 were tested for their ability to bind to the APC/C as in Fig. 2 C. Results are representative of four independent experiments. (C and D) The ΔIR and C box mutants of Cdc20 can still interact with the APC/C when part of the MCC. (C) HeLa cell line expressing inducible 3×Flag-tagged wild-type, ΔIR, C box, or ΔKILR mutant Cdc20 were treated with siRNA against Cdc20, arrested at prometaphase with 0.33 µM nocodazole, and harvested by mitotic shake off. The APC/C was immunoprecipitated using anti-APC4 antibodies and analyzed by quantitative immunoblotting. Results are representative of three independent experiments. (D) HeLa cell lines expressing inducible 3×Flag-tagged wild-type or ΔIR mutant Cdc20 were treated with siRNA against Cdc20 or Cdc20 and APC3, arrested at prometaphase, and analyzed as in C. (E and F) The ΔKILR mutant is not able to form the MCC or bind to the APC/C. (E) HeLa cell lines expressing inducible 3×Flag-Cdc20wt or Cdc20ΔKILR were treated with siRNA against Cdc20, arrested in prometaphase with 0.33 µM nocodazole, and harvested by mitotic shake off. Extracts were analyzed by size-exclusion chromatography on a Sepharose 6 column, and fractions were analyzed by quantitative immunoblotting with antibodies against Cdc20. The peaks of APC/C and MCC migration are indicated. Results are representative of two independent experiments. (F) Distributions of wild-type Cdc20 and ΔKILR mutant with the sum of Cdc20 intensities set to 1. Immunoblotting with antibodies against APC3, Cdc20, BubR1, and Mad2 is shown in Fig. S3. end, endogenous; IP, immunoprecipitation; WT, wild type.

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