Figure 5.

Effects of NEDD8 ultimate buster 1 (NUB1) overexpression on cell-cycle transition in A498 cells. A498 cells transfected with pcDNA3 (control) or pcDNA3/FLAG-NUB1 were harvested 16 h after transfection and stained with propidium iodide (PI) as described in the Materials and Methods section. (A) The percentage of cells in each phase of the cell cycle was analysed by flow cytometry. Relative fluorescence intensities in a two-dimensional fluorescence-activated cell sorting (FACS) profile (ModFit LT software) in the control (left) or NUB1-overexpressing A498 cells (right) are presented. (B) As noted in the Materials and Methods section, cells were harvested 48 h after transfection and total cell lysates were prepared and analysed by western blot analysis using antibodies directed against the indicated proteins. Each experiment was performed in triplicate. The expression levels of p27 and cyclin E were normalised by the β-actin level and presented as the relative fold compared with the control. Data values were presented as means±s.d.