Figure 1.

Ectopic UbcX^wt overrides spindle assembly checkpoint and cytostatic factor arrest in Xenopus egg extract. (A) CSF extract containing ³⁵S-securin was supplemented with nocodazole and high concentrations of sperm to activate the SAC in the CSF arrest. CSF arrest was released by the addition of calcium. Samples were taken at the indicated time points after the addition of the specified reagents, ³⁵S-securin was detected by autoradiography and XErp1 and α-tubulin by WB. (B) The indicated reagents were added to CSF extract, and the stabilities of ³⁵S-securin, XErp1 and cyclin B2 were analysed as before. (C) The extract was treated as in (B) and chromatin structures were analysed after 90 mins. (D) The amount of UbcX in CSF extract was determined by WB, using recombinant UbcX^wt as standard. (E) CSF extract was supplemented with the indicated concentrations of UbcX^wt and CSF release was monitored by autoradiographic analysis of ³⁵S-securin. (F) Stage VI oocytes were injected with buffer or with 80 ng UbcX^wt, resulting in 8.9 μM exogenous UbcX, which is approximately 11-fold more than levels of the endogenous protein. Maturation was induced by progesterone treatment and samples were taken for WB analysis at the indicated time points after GVBD. ci, catalytically inactive; CSF, cytostatic factor; GVBD, germinal vesicle breakdown; PG, progesterone; SAC, spindle assembly checkpoint; ³⁵S-securin, in vitro translated ³⁵S-labelled securin; WB, western blotting; wt, wild type.