Fig. 5.

Phosphorylation of Hbo1 by Plk1 is essential for pre-RC complex formation. HeLa cells were cotransfected with Flag-Hbo1 (A and B) or pBS/U6-Plk1 (C) and pBabe-puro at a ratio of 7:1 and selected with puromycin. After floating cells were washed away, attached cells were further incubated for 12 h and then harvested. To monitor the Plk1-depletion efficiency, attached cells were treated with nocodazole for 14 h and then harvested (lower portion of C). Lysates were separated into cytoplasmic, soluble nucleoplasmic, and chromatin-enriched fractions and then subjected to Western blot using antibodies indicated at left. The effect of Plk1 depletion on Mcm loading onto chromatin is shown in the upper portion of C.