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Fig. 7. Meiosis II degradation of cdc20 is KEN-box dependent. (A) Wild-type cdc20 coupled to GFP was expressed in MII eggs that were subsequently inseminated. Following sperm-triggered Ca²⁺ spiking cdc20 degradation was observed around the time of second polar body extrusion and continued until pronuclei formed in the 1-cell embryo. (B) Degradation of cdc20 was observed to be dependent on the presence of a KEN box, since its removal rendered cdc20 stable. Thus despite fertilization of these eggs, as observed by a series of Ca²⁺ spikes, cdc20^km:: GFP levels continued to rise throughout the completion of meiosis II. Recordings are representative of 8 eggs for both constructs. (C) Endogenous cdc20 was observed to be degraded at the two time points sampled: 2 hours (+2hr) and 6 hours (+6hr) after addition of Sr²⁺-containing medium to induce parthenogenetic activation. Polar bodies were extruded after about 1 hour, and pronuclei were visible at around 5 hours. (i) western blot; (ii) Coomassie Blue stained membrane; (iii) eggs at time 0 hours and 6 hours probed for cdc20 and actin as a loading control. All lanes were loaded with 50 eggs.

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