Figure 4.

Overexpression of K6W-ubiquitin inhibits the degradation of mitotic regulators. HLEC were synchronized and infected with adenovirus as described in Figure 3. After replating, the cells were collected at the indicated times. Levels of cyclin A, cyclin B, and securin were determined by western blotting using polyclonal antibodies to cyclin A and securin, and monoclonal antibody to cyclin B (A). B-D: The densitometry quantifications of cyclin A, cyclin B, and securin, respectively, after normalization for protein loading by measuring levels of ubiquitin-conjugating enzyme E1, which remains a constant level during cell cycle. Experiments were performed three times with reproducible results. The asterisk indicates a p<0.05 and the double asterisk indicates a p<0.01 as comparing cells infected with adenovirus encoding K6W-ubiquitin with cells infected with empty adenovirus.