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Fig. 1. Western blot assay of nuclear extracts (NE) from wild-type (HCT116) and Ku80^+/- cells. NE were prepared from HCT116 and Ku80^+/- by high-salt extraction of isolated nuclei and were immunoblotted for both subunits of Ku. The expression of p53 and p21 was examined to determine whether they were induced by the reduced Ku80 levels in Ku80^+/- cells. Expression of Cdc6 and Cdk2 were also inspected to verify that Ku80 deficiency did not reduce their expression. Nuclear actin was used as a loading control. (A) Increasing amounts (10, 20 and 30 µg) of NE were subjected to electrophoresis and western blotting (as described in Materials and Methods). Immunoblotting with 1/100^th dilution of anti-Ku80 (C-20), or 1/1000^th dilution of anti-Ku70 (C-19), anti-p53 (FL393), anti-p21 (C19), anti-Cdc6 (D12), anti-Cdk2 (H304), and anti-actin antibody was carried out. (B) Histogram plots of quantifications of the Ku80, Ku70 and actin bands shown in A. Each error bar representing three experiments and one standard deviation (s.d.) is indicated. Signals obtained for HCT116 NE were set at 100%, and those of Ku80^+/- were expressed as a percentage of them. An asterisk (^*) represents statistically significant differences (P<0.05) in the expression of the indicated protein between HCT116 and Ku80^+/- cells.

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