Fig. 3.

Protection of sites affecting the binding of Cdc20 to APC/C against phosphorylation by Cdk1. (A) Protection against Cdk1 action of a mutant of Cdc20 (5AV) that can be phosphorylated only on three T residues at its N-terminal region. Free or APC/C-bound forms of the indicated wild-type or mutant myc-Cdc20s were incubated (at 23 °C for 30 min) with or without Cdk1, as described for Fig. 2A. Myc-Cdc20 bound to APC/C was detected by immunoblotting and was expressed as the percentage of the control incubated without Cdk1. In separate samples of immunoprecipitates, not treated by lambda phosphatase, the activity of APC/C was determined as described for SI Appendix, Figs. S1 A and B and results were expressed as described for Fig. 2C. (B) Patterns of phosphorylation of free and APC/C-bound 5AV mutant of Cdc20, analyzed by Phos-tag-SDS-PAGE. Free of APC/C-bound mutant of myc-Cdc20 were subjected to phosphorylation by Cdk1, as described in Fig. 2 A and D. Samples were resolved by Phos-tag-SDS-PAGE and were immunoblotted with Myc-tag. Phosphorylated free myc-Cdc20 5AV was applied at one-half (lane 3, “0.5”) or at equal amount (lane 4, “1.0”) to that of wild-type myc-Cdc20 (lane 5).