Cooperative inhibition of RIP1-mediated NF-κB activation by UL45 and UL48 in reporter assays.

<p>(A) 293T cells in six-well plates were co-transfected using PEI with 0.1 μg of NF-κB-luciferase and 0.5 μg of SV2-β-galactosidase (for normalization) reporter plasmids, and 0.5 μg plasmids for Myc-UL45 and HA-RIP1 as indicated. At 24 h after transfection, total cell lysates were prepared and assayed for luciferase activity. (B and C) HF cells in six-well plates were co-transfected using OmicsFect with 0.5 μg of NF-κB-luciferase and 0.5 μg of SV2-β-galactosidase reporter plasmids and 1 μg plasmid for HA-UL45 or 0.5 μg HA-RIP1. Plasmid (0.5 μg) for CrmA was added in (B) and TNFα (50 ng/ml) was added for 8 h before cell harvest in (C). (D) 293T cells were co-transfected using PEI with NF-κB-luciferase and 0.5 μg of SV2-β-galactosidase reporter plasmids and 0.1 μg of plasmids for Myc-UL45 and Myc-UL48 (wild-type or C24S mutant), or 0.5 μg of HA-RIP1. (E and F) HF cells were co-transfected using electroporation with 0.5 μg of NF-κB-luciferase reporter plasmid, SV2-β-galactosidase reporter plasmid, and plasmids for Myc-UL45, Myc-UL48 (wild-type or C24S mutant), HA-RIP1, and CrmA, as indicated. Total cell lysates were prepared 24 h after transfection and assayed for luciferase activity. TNFα (50 ng/ml) was treated for 8 h before cell harvest in (F). Luciferase activity was normalized with β-galactosidase activity. Shown are mean values with the standard errors of luciferase activity of three independent assays.