(A) Purified unlabeled Cdc20 was pre-incubated with 0.5 μM Mad2 and the indicated Mad3-Bub3 concentration. Reactions were started by the addition of purified 35S-securin, APC/C (5 nM), and E1/E2(Ubc4)/methyl-ubiquitin mix. Similar results were obtained in three independent experiments.
(B) Purified 35S-Cdc20 was pre-incubated with 5 μM Mad2 and the indicated Mad3-Bub3 concentration. Reactions were started by the addition of APC/C (5 nM) and E1/E2(Ubc4)/methyl-ubiquitin mix. Similar results were obtained in two independent experiments.
(C) The data from (A) and (B) were analyzed using Prism, and the zero concentration was plotted on a log scale as 0.01 nM.