FIGURE 5.

Analysis of the effect of viral NP on p53 stability. Vero cells were transiently transfected with plasmid Flag-NP, Flag-M1, Flag-NS2, Flag-NS1, Flag-M2 or Flag-vector (Vec) in the presence of p53-Luc reporter plasmid and incubated for 24 h. A, protein expression was detected by Western blot analysis using the indicated antibodies. B, p53 luciferase activity of lysates prepared from the transfectants was analyzed. Results are presented as the means ± S.E. from three independent experiments. *, p < 0.05 compared with cells transfected with the empty vector. C, Vero cells were transiently transfected with plasmid Flag-NP or Flag-vector and incubated for 24 h. The transfectants were treated with CHX at 100 μg/ml for the indicated times (hours) post-treatment (hpt) and subjected to Western blot analysis using the indicated antibodies. D, Vero cells were transiently transfected with plasmid Flag-NP or Flag-vector (Vec) and incubated for 24 h. The transfectants were treated with 10 μm of MG132 or mock-treated for 6 h and subjected to Western blot analysis using the indicated antibodies. To detect the ladders of polyubiquitinated p53 (Ub-p53), films were exposed for longer. E, change in abundance of polyubiquitinated p53 (Ub-p53) in the transfectants was determined by densitometric analysis and normalized to β-actin. The relative abundance of Ub-p53 was plotted. Results are presented as the means ± S.E. from three independent experiments. *, p < 0.05 compared with cells transfected with Flag-vector.