FIG 5.

Effect of UL50 proteins on VCP/p97 expression in transfection assays. (A and B) 293 cells in six-well plates were cotransfected with plasmids (1 μg) expressing Flag-VCP/p97 or UL50-HA (wild type or mutants), as indicated. At 24 h after transfection, cell lysates were prepared and immunoblot assays were performed with anti-Flag or anti-HA antibodies. The levels of β-actin, which was used as a loading control, are shown. Relative Flag-VCP/p97 levels normalized to the levels of β-actin are indicated. (C) HF cells in 12-well plates (1.5 × 10⁵ cells per well) were mock infected or infected with increasing amounts (3 and 6 50% tissue culture infective doses per cell) of recombinant adenoviruses expressing UL50(M199V)-HA or β-galactosidase (β-Gal.; LacZ), as indicated. Cells were harvested at 48 h after infection, and immunoblotting was performed with anti-VCP/p97, anti-HA, or anti-β-actin antibodies. Expression of β-galactosidase was determined by Coomassie blue staining. Transcript levels of VCP/p97 were also measured by RT-quantitative PCR. VCP/p97 mRNA levels normalized to those of β-actin are shown as a graph. (D) HF cells in 12-well plates (1.5 × 10⁵ cells per well) were infected with recombinant adenoviruses, as described in the legend to panel C, at an MOI of 3. Cells were treated with sterile dimethyl sulfoxide (lane D), MG132 (20 μM; lane M), or bafilomycin A1 (100 nM; lane B) for 4 h, as indicated, prior to cell harvest at 60 h after infection. Cell lysates were prepared and subjected to immunoblotting with antibodies to VCP/p97, HA, UBE1L, LC3, or β-actin.