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Fig. 3. Origin activation and DNA synthesis as a function of Ku80 knockdown. (A) Maps of the lamin B2 (left) and c-myc (right) origin loci, and melting curves of the primers corresponding to the origin-containing (LB2 and Myc11, respectively) and origin-lacking (LB2C and Myc1, respectively) amplicons. The location of the amplicons relative to the gene exons as well as their distance in kb is indicated on the maps. (B,C) Histogram plots of the lamin B2 and c-myc origin activities throughout the silencing time-course, as measured by nascent-DNA-strand abundance. The LB2 and Myc11 regions lie within the origins, whereas LB2C and Myc1 are distal origin-lacking regions. Values are expressed as ng of nascent DNA and represent three experiments ± 1 s.d. (D) Representative western blot analysis of PCNA protein expression at 72 hours post-transfection with Ku80 siRNA. Actin was used as a loading control. (E) Representative western blot analysis of the phosphorylation levels of the H2AX histone variant at 96 hours of Ku80 RNAi. As positive controls, extracts from cells treated with the drugs hydroxyurea (inhibitor of ribonucleotide reductase) and nocodazole (inhibitor of microtubule formation) were used. Immunostaining with a PanH3 antibody was used as a loading control.
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