Figure 6.

Expression of D2 uncouples the activation of p42^mpk1 and pre-MPF in response to progesterone. (A) Groups of 30 oocytes were injected with water or mRNAs encoding either D1 or D2 and after overnight incubation were treated with progesterone. Every hour, starting 3 h after the addition of progesterone, two or three progesterone-treated oocytes without a white spot were transferred to dry ice. Untreated oocytes (control) and progesterone-treated oocytes that had a white spot were also taken for comparison. Oocyte lysates were prepared from single oocytes and analyzed by immunoblotting with anti-p42^mpk1 and anti-p34^cdc2 antibodies. Each bar represents a single oocyte. A total of 30 oocytes were analyzed in three independent experiments. The activation of p42^mpk1 was scored by the phosphorylation of the protein, which causes its upward mobility shift in the blots. The activation of pre-MPF was scored for the disappearance in the blots of the slow migrating p34^cdc2 form, which corresponds to Tyr-phosphorylated cyclin B-bound p34^cdc2. (B) Representative examples of the p42^mpk1 and p34^cdc2 immunoblots from single oocytes described in A.