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Figure 4


Fig. 4. Expression and chromatin loading of DNA replication licensing factors. (A) Representative western blot analyses of Ku80, Orc1, Orc2, Orc3, Orc4, Orc6, MCM7 and Cdc45 protein levels in whole cell extracts prepared from untreated, control, scr-siRNA-transfected and Ku80-siRNA-transfected HeLa cells at 72 hours post-transfection. Actin was used as a loading control. (B) Relative optical densities (ROD) of Ku80, Orc1, Orc2, Orc3, Orc4, Orc6, MCM7 and Cdc45 immunoreactivities. Values are expressed as ratios of the individual RODs to the one corresponding to untreated cells, and the error bars represent 1 s.d. of three experiments. (C) Representative western blot analyses of Ku80, Orc1, Orc2, Orc3, Orc4, Orc6, MCM7 and Cdc45 protein levels in the chromatin-bound fraction prepared from untreated, control, scr-siRNA-transfected and Ku80-siRNA-transfected HeLa cells 72 hours post-transfection. Orc2, the association of which with chromatin is essentially the same throughout the cell cycle, was used as loading control. (D) Quantification of the immunoreactivity of the proteins shown in C after Ku80 knockdown for 72 hours. Signal intensities (RODs) are means ± 1 s.d.





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