Cdt2 readily promotes Xic2 turnover in the presence of p27 CDK inhibitor. A. GST pull-down assay. GST alone or GST fused to wildtype Xic1 (Xic1WT), I174A mutant of Xic1 (Xic1I174A), Xic2, p21, or p27 (shown in coomassie gel shown in bottom panel) were bound to glutathione sepharose and incubated with in vitro translated 35S-methionine labeled Xenopus Cdt2 (XCdt2). 5% of the input XCdt2 is shown in lane 1 of the top panel (5% Input). B. Xic2 degradation assay. Top panel: 35S-methionine labeled Xic2 was incubated in HSS with (+) or without (-) 10 ng/ul ssDNA (ssDNA) in the presence (+) or absence (-) of p27, unprogrammed reticulocyte lysate (Unprog), or unlabeled in vitro translated Cdt2 (CDT2) for 0 to 3 hrs as indicated. Bottom panel: Graphic representation of Xic2 degradation. 35S-methionine labeled Xic2 was incubated in HSS with 10 ng/ul ssDNA and the percentage of Xic2 remaining was calculated for each sample where the zero hour time point was normalized to 100%. Reactions were supplemented with unprogrammed reticulocyte lysate (Unprog) (7 experiments), unlabeled in vitro translated Cdt2 (Cdt2) (7 experiments), unprogrammed reticulocyte lysate with p27 (Unprog+p27) (4 experiments), unlabeled in vitro translated Cdt2 with p27 (Cdt2+p27) (4 experiments), or unlabeled in vitro translated Xenopus Skp2 (Skp2) (3 experiments). Error bars (Standard error of the mean) are shown. P values were calculated by student t-test comparing each sample with the addition of unprogrammed reticulocyte lysate (Unprog). 1.5 hr p values: Cdt2 (0.000463), Unprog+p27 (0.270), Cdt2+p27 (0.00184), Skp2 (0.702). 3 hr p values: Cdt2 (0.00120), Unprog+p27 (0.0130), Cdt2+p27 (6.56E-05), Skp2 (0.306).