Figure 4

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 (Xic1^WT), I174A mutant of Xic1 (Xic1^I174A), 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).