Fig. 2.
Substrate degradation in Xenopus egg extracts induced by the addition of WT, pA, or pE. pA, pE, or WT was added back to APC/C-depleted egg extract and incubated with nondegradable Δ90 cyclin B1 for 90 min. Substrates were added, and samples were taken and analyzed at the indicated time points for CycBNTD (cyclin B1 residues 1–87) and securin degradation by Western blotting. Equal amounts of each APC/C variant were used (see APC3 Western blot). SMC3 was used as a loading control. (A) pA vs. WT in mitotic egg extract. (B) pA vs. WT in interphase egg extract supplemented with recombinant human CDH1 protein. (C) pE vs. WT in interphase and mitotic egg extracts. (D) pE in APC/C or APC/C- and CDC20-depleted interphase and mitotic egg extracts supplemented with recombinant human CDC20. (E) Securin degradation time course in mock-depleted interphase (blue) and mitotic (yellow) egg extracts and APC/C-depleted egg extracts following add-back of WT in interphase (gray) or mitosis (red) or add-back of pE in interphase (orange) or mitosis (green). Securin levels were monitored by Western blotting and normalized to ORC2 levels. SEM, n ≥ 2.