Phosphorylated Cyclin E protein is degraded by xCdc4. (A) Two-cell-stage embryos were injected with 1 ng of amino-terminally FLAG-tagged Cyclin E mRNA (lanes 2 to 5) and either 1 ng of GFP mRNA (lane 2) or 1 ng of xCdc4 mRNA (lanes 3 to 5). The ability of xCdc4ΔFbox to block Cyclin E degradation was examined by co-injecting 1.5 ng of xCdc4ΔFbox mRNA (lane 5), with 1.5 ng of GFP mRNA or 1.5 ng of Skp2ΔFbox mRNA as controls (lanes 3 and 4). Embryos were allowed to develop to stage 10.5 and immunoblotting was performed for the FLAG epitope, while immunoblotting of actin was performed as a loading control. IR-Dye™-conjugated secondary antibodies were used, enabling accurate quantification of Cyclin E and actin expression. (B) The level of phospho-Cyclin E was normalized to the level of actin, then compared to the level of phospho-Cyclin E in GFP-injected embryos, showing mean ± standard error of the mean from four independent experiments.