Fig. 2. The degradation of Aurora-A is mediated by the APC/Fizzy-Related complex and is blocked by the addition of a cyclin B D-Box-motif-containing peptide. (A) An interphase extract (50 µl) was first depleted with either α-CDC27 antibodies (α-CDC27 IP) or non-specific antibodies (Control IP) and then complemented with Fizzy-Related mRNA. A sample of 2 µl was then taken at different times, and endogenous Aurora-A levels were analysed by western blotting. The initial interphase extract (CDC27, Int) and the supernatants of α-CDC27 (CDC27, SN Cdc 27) and non-specific (CDC27, SN Ct) immunoprecipitations were analysed by western blotting with α-CDC27 antibodies to verify CDC27 depletion. (B) Fizzy-Related mRNA was added to interphase extracts (50 µl); 20 min later, we added a purified recombinant cyclin A protein (2 ng/µl, +Cyclin A), the cyclin B D-Box-containing peptide (7 µg/µl, +DBox Peptide) or buffer (CT). Endogenous levels of Aurora-A were then analysed by western blotting at different times.