Fig. 3. Human Aur-A IVT product is destroyed in egg extract without additional Cdh1. (A,B) Xenopus egg CSF extract was incubated for 5 minutes with the APC/C activators Cdc20 or Cdh1. Radiolabeled IVT substrates were added as indicated and incubated for a further 10 minutes. Calcium was added to trigger exit from meiosis-I metaphase arrest and entry into the first cell cycle. Samples were taken at the indicated times and analysed by PAGE and autoradiography. (C) Radiolabeled IVT products were prepared, using 40 µg µl^-1 DNA template or 20 µg ml^-1 IVT and purified mRNA template. Templates were: (1) Xenopus Aur-A DNA; (2) human Aur-A DNA; (3) Xenopus Aur-A mRNA; (4) human Aur-A mRNA; (5) human Aur-A D-box-mutant DNA; (6) human Aur-A (66-403) DNA; and (7) human Aur-A DNA in the presence of 10 ng µl^-1 Cdh1(1-125) protein. (D) Following coupled in vitro transcription-translation of the indicated templates for 90 minutes in rabbit reticulocyte lysate, 100 µg ml^-1 cycloheximide (CHX) was added to inhibit further protein synthesis. Reaction mixes were shifted to 23°C and samples were taken at subsequent times for analysis by SDS-PAGE followed by autoradiography. Where indicated, the proteasome inhibitor MG-115 (1.6 µM) was added 10 minutes before CHX.