FIG. 3.

The effect of inhibiting the Paf/PI3 kinase/AKT pathway on TRP53 and pMDM2 expression in zygotes cultured for 90 h in vitro. A) Hybrid zygotes cultured for 96 h without treatment; embryos lacking the Paf-receptor (Ptafr^−/−); B6CBF2 zygotes cultured in an inhibitor of PI3 kinase (LY294002, 15 μM); or B6CBF2 zygotes cultured in an inhibitor of AKT (AKT inhibitor 10 μM). Images are single equatorial confocal sections of representative embryos from three replicate experiments. Magnification ×450. B) Mouse zygotes were cultured overnight in media with or without Paf. The total amount of pMDM2 staining in the resulting 2-cell embryos was assessed by Western blot analysis. pMDM2 Western blot analysis from three replicates are shown, and the staining for actin from the same gel is also shown for replicate 3 (Rep3). Densitometric analysis (mean + SEM) of the Western blots are also shown. AU, arbitrary units, modHTF, modified HTF. C) In an experiment of similar design as B, embryos cultured in the presence of Paf were treated with a PI3-kinase inhibitor (LY294002) or an AKT inhibitor (deguelin) and the amount of pMDM2 assessed by Western blot analysis. A representative Western blot is shown and the densitiometric analysis (mean + SEM) of four such blots is presented in the graph.