MAD2L2 degradation in anaphase is mediated by APC/C^CDC20. (A) D-box and proteasome-dependent destruction of MAD2L2 in extracts of mitotic Xenopus oocytes. The first 141 amino acids of MAD2L2, and the first 200 amino acids of Cyclin B1, were fused to luciferase, in vitro translated, and added to mitotic Xenopus oocyte extracts. Degradation is stabilized by mutation of the D-box or by addition of MG132. The graphs shows quantification of three repeats of the experiment with the level of translated MAD2L2 being normalized to time 0. Error bars = 1 SD. (B) The degradation of MAD2L2 in early anaphase is delayed by depletion of CDC20. Depletion of CDC20 alone results in prolonged delay in mitotic exit and hence also a delay in MAD2L2 and Cyclin B1 degradation and CDC27 dephosphorylation. Addition of the CDK1 inhibitor RO3306 permits mitotic exit in the absence of CDC20, but MAD2L2 degradation remains delayed.