Figure 5: The APC/C–Cdc20 is active in interphase.

(a) Stable HeLa cell lines expressing the indicated YFP-tagged Cdc20 proteins were depleted of endogenous Cdc20, released from one 20-h thymidine block and cells were collected 9 h after the release. Samples were analysed by SDS–PAGE and the level of the indicated proteins was determined by quantitative western blot. (b) Quantification of six experiments similar to a. Western blot fluorescence intensity signal was normalized to either Vinculin or GAPDH and the level in YFP–Cdc20^WT-expressing cells was set to 1. The average and s.d. is indicated. (c) Outline of synchronization and experimental set-up to look at protein turnover. (d) Cells were depleted of endogenous Cdc20, released from a 20-h thymidine block and 6 h after release treated with cycloheximide (CHX) and with MG132 where indicated (+) for 80 min. Total cell extract was analysed by western blot and probed for the indicated proteins. (e) Quantification of five experiments as in d. Western blot fluorescence intensity signal was normalized to GAPDH and Cdc20^WT set to 1. The bar diagram shows the average and s.d.