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. 2020 Jun 16;94(13):e00110-20. doi: 10.1128/JVI.00110-20

FIG 6.

FIG 6

Effect of UL50-p26 on pUL50-mediated VCP/p97 loss and IE1/IE2 downregulation. (A) 293 cells in six-well plates were cotransfected with the pRL45 plasmid containing the MIE gene (0.5 μg), a plasmid expressing Flag-VCP/p97 (0.5 μg), and a plasmid (0.5 μg) containing the UL50-HA gene (wild type or mutants), as indicated. At 24 h after cotransfection, cell lysates were prepared and immunoblotting analysis was performed with anti-810R (for IE1/IE2), anti-HA, anti-Flag, or anti-β-actin (as a loading control) antibodies. Relative IE2/IE1 ratios are indicated. (B) 293 cells in six-well plates were cotransfected with the pRL45 plasmid (0.25 μg), a plasmid (0.5 μg) expressing Flag-VCP/p97 or pUL50 [UL50(M199V)-HA], and increasing amounts (0.125 or 0.25 μg) of a plasmid expressing UL50-p26-HA [UL50(199–397)-HA], as indicated. At 24 h after cotransfection, cell lysates were prepared and immunoblotting analysis was performed as described in the legend to panel A.