Fig. 4.

The overexpression of Hbo1-S57A leads to G₁/S arrest in HeLa cells. (A and B) Cells were cotransfected with Flag-Hbo1 or Flag-Hbo1-S57A and pBabe-puro at a ratio of 7:1 and selected with puromycin. After floating cells were removed, attached cells were further incubated for 10 h in the presence or absence of nocodazole. Lysates were prepared and subjected to Western blot analysis (A) or an anti-Cdk1 IP/kinase assay using histone H1 as a substrate (B). (C) After transfection as in A, cells were incubated for 6 h, treated with nocodazole for 5 h, and harvested for FACS analysis. (D) At 3 d after transfection with GFP-Hbo1 or -S57A, cells were labeled with BrdU for 30 min and stained with BrdU antibody, and the GFP-positive cells were counted. (E and F) Cells were cotransfected with Hbo1 constructs and pBabe-puro at a ratio of 7:1 (E) or with pBS/U6-Plk1, Flag-Hbo1, and pBabe-puro at a ratio of 6:3:1 (F), selected with puromycin, and treated with nocodazole. Nuclear extracts were prepared and subjected to a HAT activity assay.