FIG. 9.

Lack of an effect of 3-MA or the overexpression of ubiquitin on MG132 inhibition of late gene expression. (A) Effect of 3-MA. Following adsorption with vpF17R-CAT, HeLa cells were incubated with DMSO, 3-MA, MG132, or 3-MA plus MG132. CAT activity was determined 8 h after infection. The values were normalized to the activity with no inhibitor. (B) CAT expression after the overexpression of ubiquitin. HeLa cells were transfected with the indicated amounts of plasmid encoding hemagglutinin-tagged ubiquitin (pHA-Ub) regulated by the cytomegalovirus early promoter. After 24 h, the cells were inoculated with vpF17R-CAT and then incubated in the presence of DMSO or 10 μM MG132. CAT activity was determined 8 h after infection and normalized. (C) Modification of proteins by epitope-tagged ubiquitin. HeLa cells were transfected as described for panel B. After 24 h, the cells were treated with DMSO or 10 μM MG132 for an additional 8 h. The lysates were analyzed by Western blotting with antibody to the influenza virus epitope tag. The position of free ubiquitin is shown on the left, and the positions of the marker proteins are on the right.