FIG. 4.

Vpr-binding domain located between aa 291 and 369 in Wee1 is required for the dominant-negative effect. (A) Constructs of Wee1 deletion mutants (left panel). Dark boxes represent a part of the Wee1-kinase domain located from aa 291 to 575. The light gray box represents the Vpr-binding domain located from aa 291 to 369. All mutants contain an HA tag at the N terminus and a Myc tag at the C terminus. Wee1 deletion mutants and EGFP-encoding plasmids were introduced into HeLa cells by nucleofection according to the manufacturer's instructions. The expression of Wee1 deletion mutants was analyzed by Western blotting with anti-HA specific antibody. β-Tubulin was the loading control (right panel). (B) Cells were synchronized at G₁/S by a double-thymidine block and then infected with lentiviral vector encoding either Flag-tagged Vpr (Vpr) or EGFP (as a control) at two different MOIs (400 and 600 ng of viral p24 antigen per 10⁵ cells), or gamma irradiated at 4,000 rads. Cells were then stained with propidium iodide at 12 h postinfection and analyzed by flow cytometry. A total of 10,000 events were collected and analyzed per sample. The number in each panel indicates the percentage of cells in each peak.