Figure 4. CycB-NT promotes Cdc20 binding to the APC and suppresses Cdc20 ubiquitination.
(a) Cyclin B-NT increases the steady-state level of Cdc20 bound to the APC in non-treated or TAME-treated mitotic Xenopus extract. A cyclin B1 N-terminal fragment (CycB-NT, 6 μM) or the same fragment without the D-box (CycB-NT ΔD-box, 6 μM) and TAME (200 μM) were added to mitotic extract as indicated. APC was immunoprecipitated from the extract and protein levels were analyzed by Western blot. (b) CycB-NT promotes Cdc20 binding to the APC in the absence of the IR-tail. APC cleared of Cdc20 by TAME-treatment was incubated with in vitro-translated WT Cdc20 or Cdc20ΔIR for 10 minutes. CycB-NT (2 μM) and TAME (200 μM) were added as indicated. (c) CycB-NT suppresses Cdc20 ubiquitination. The same assay was performed as in Fig. 2b in the presence or absence of 500 nM cycB-NT.