Figure 3

XCdc14α is mitotically phosphorylated during oocyte maturation and in the Xenopus embryonic cell cycle. A. XCdc14α protein levels are constant during development. Xenopus eggs were fertilized in vitro and at the indicated stages were frozen down for subsequent immunoblot analysis. B. XCdc14α is phosphorylated in mitosis in cycling Xenopus extracts. At the indicated times, samples from cycling extracts were frozen in liquid nitrogen for subsequent immunoblot analysis with anti-XCdc14α antibodies (top panel) and total histone H1 kinase activity (lower panel). For the immunoblot, lysates were resolved on 11% SDS PAGE gels. C. XCdc14α becomes phosphorylated after germinal vesicle breakdown (GVBD) in the maturing oocyte. Defolliculated Stage VI oocytes were induced to mature by the addition of progesterone (10 μg/ml). At the indicated times, oocytes were flash frozen for subsequent immunoblot analysis with antibodies against XCdc14α. GVBD occurred between 2 and 2.5 hrs. D. The majority of XCdc14α is monomeric or dimeric through the cell cycle. At 0, 70 and 90 min, samples from cycling extract were flash frozen in liquid nitrogen. Left: samples from the cycling extract were tested for total histone H1 kinase activity. Arrows indicate timepoints that were analyzed. Right: The frozen samples were diluted 10-fold in XB buffer, and 50 μg (1 μl equivalent of undiluted lysate) was fractionated on a Superdex200 gel filtration and probed with anti-XCdc14α antibodies. The elution profiles of molecular weight standards are depicted above the fractions.