FIGURE 6.

Analysis of the effect of viral NP on Mdm2-mediated p53 ubiquitination. H1299 cells were transiently transfected with a combination of the indicated plasmids and incubated for 24 h. The transfectants were treated with 15 μm MG132 for 3 h before harvest. A, ubiquitinated p53 (Ub-p53) in the transfectants was pulled down using Ni²⁺-NTA-agarose beads and detected by Western blot analysis with anti-p53 antibody (DO-1) (panel a). The protein expression in the transfectants was detected by Western blot analysis using the indicated antibodies (input panels). B, change in abundance of Ub-p53 in the transfectants was determined by densitometric analysis. The ratio between ubiquitinated and non-ubiquitinated p53 (Ub-p53/p53) was plotted. Results are presented as the means ± S.E. from three independent experiments. *, p < 0.05, **, p < 0.01 compared with cells transfected with pcDNA-p53, Flag-Mdm2, and His-ubiquitin.