FIGURE 8.

Effect of RNF4 on the stability and transactivation activity of Rta. A, 293T cells were transfected with plasmids pCMV-R and control shRNA (Ct-shRNA) or RNF4 shRNA. Cycloheximide (CHX) was added at 40 h after transfection to inhibit protein synthesis. Whole cell lysate was prepared at 0, 30, 90, and 150 min after transfection. Proteins in the lysate were detected by immunoblotting (IB) using anti-Rta, anti-α-tubulin, and anti-RNF4 antibodies. B, a densitometric analysis of Rta level normalized to α-tubulin was plotted using Image J software. Data are presented as the mean with S.D. and represent three independent experiments. The intensity corresponding to 50% of the initial value is indicated by the horizontal line. C, 293T cells were cotransfected with the reporter plasmid pBMLF1 and pCMV-R, pEGFP-RNF4, or pEGFP-RNF4-CS1. Luciferase activities were monitored 24 h post-transfection. Each transfection experiment was performed three times, and each sample in the experiment was prepared in duplicate. The value from each experiment was analyzed statistically with the least square means method. *, p < 0.05.