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. 2022 Aug 24;119(35):e2210367119. doi: 10.1073/pnas.2210367119

Fig. 2.

Fig. 2.

Differences in effects of Cdk1 on free and APC/C-bound Cdc20. (A) Effects of Cdk1-catalyzed phosphorylation of free or APC/C-bound Cdc20 on its association with APC/C. (Upper) Free myc-Cdc20 (50 nM) was phosphorylated in a reaction mixture that contained in a volume of 50 mL: 40 mM Tris-HCl (pH 7.6), 3 mM MgCl2, 1 mM ATP, 10 mM phosphocreatine, 0.1 mg/mL creatine phosphokinase, 2 mg/mL BSA and 40 units/mL Cdk1-cyclin B (referred to as “Cdk1”). Following incubation at 23 °C for the time periods indicated, the binding of myc-Cdc20 to immunopurified APC/C (on 4 mL anti-Cdc27 beads) was assayed following washing of beads as described under Materials and Methods. (Lower) myc-Cdc20 pre-bound to immunopurified APC/C (see Materials and Methods) was incubated with Cdk1 under conditions similar to those described above and then beads were washed and the amount myc-Cdc20 that remained associated with APC/C was estimated by immunoblotting. All samples had been subjected to treatment with lambda phosphatase. “No APC/C,” nonspecific absorption of myc-Cdc20 to anti-Cdc27 beads without APC; “No Cdk1,” samples incubated without Cdk1 for 30 min. (B) Quantitation of data from A. Background values (“No APC/C”) were subtracted and myc-Cdc20 bound to APC/C were expressed as the percentage of the control sample incubated without Cdk1. (C) Effects of phosphorylation by Cdk1 of free or APC/C-bound myc-Cdc20 on the activity of APC/C. Quantitation of APC/C activity data from SI Appendix, Figs. S1 A and B. Background values if ubiquitylated 125I-cyclin obtained without added myc-Cdc20 (lanes 2 in SI Appendix, Figs. S1 A and B, “No Cdc20”) were subtracted and results were expressed as the percentage of sample incubated without Cdk1 for 30 min (lane 3 in SI Appendix, Fig. S1A and lane 4 in SI Appendix, Fig. S1B). (D) Phos-tag-SDS-PAGE analysis of patterns of phosphorylation by Cdk1 of free and APC/C-bound myc-Cdc20. The experiment was similar to that described in A, except free myc-Cdc20 was not applied to APC/C, samples were not treated with lambda phosphatase and were subjected to Phos-tag-SDS-PAGE (see Materials and Methods).