Figure 2.

UbcH10^wt-induced cytostatic factor release requires anaphase-promoting complex/cyclosome activity. (A) Cdc20 was depleted from CSF extracts by three rounds of immunodepletion. Cdc20 was detected by WB analysis in the input fraction, in the extract after the third round of Cdc20 depletion (ΔCdc20) or control depletion, as well as on the α-Cdc20 and control beads (IP) after the indicated rounds of depletion. (B) Cdc20- or control-depleted extract was supplemented with calcium, buffer, UbcH10^wt or UbcH10^ci and the stability of ³⁵S-securin was analysed by autoradiography. (C) CSF extract that was depleted of Cdc27 or control-depleted extract was supplemented with the indicated reagents and ³⁵S-securin was detected at the indicated time points. APC/C, anaphase-promoting complex/cyclosome; Cdc, cell division cycle; ci, catalytically inactive; CSF, cytostatic factor; Ctrl, control; IgG, immunoglobulin G; IP, immunoprecipitation; ³⁵S-securin, in vitro translated ³⁵S-labelled securin; WB, western blotting; wt, wild type.