CADM1 is required for KSHV vFLIP to activate NF-κB.

<p>(A) NF-κB luciferase assay using lysates of HeLa cells expressing increasing amounts of CADM1 or vFLIP. HeLa cells were transfected with increasing amounts of either CADM1 or vFLIP with κB‐TATA Luc and pRL‐tk plasmids. After 36 hours, lysates were subjected to dual luciferase assays. The lysates were also subjected to immunoblotting to examine CADM1 and vFLIP expression using anti-Flag antibody. (B) NF-κB luciferase assay using lysates of HeLa cells stably expressing control scrambled shRNA or three different CADM1 shRNAs and transfected with pRL-tk, κB-TATA Luc and vFLIP as indicated. Immunoblot analyses of CADM1 protein expression in HeLa cells after transduction with lentiviruses expressing different shRNAs targeting distinct sequences of the CADM1 transcript. (C) NF-κB luciferase assay using lysates of <i>Cadm1</i>^<i>+/+</i> and <i>Cadm1</i>^{<i>−/−</i>} MEFs transfected with pRL-tk internal control Renilla luciferase plasmid, κB-TATA Luc and vFLIP as indicated. The lysates were also subjected to immunoblotting to examine vFLIP expression. (D) A schematic overview of the FLAG-CADM1 deletion mutants ΔSP, ΔCP, ΔEC, ΔPDZ-BM and ΔFERM. (E) NF-κB luciferase assay of lysates of <i>Cadm1</i>^{<i>−/−</i>} MEFs transfected with an NF-κB firefly luciferase reporter and a renilla luciferase vector reporter together with empty vector or an expression vector for Flag-tagged wild-type CADM1, CADM1 ΔSP, CADM1 ΔCP, CADM1 ΔEC, CADM1 ΔPDZ-BM and CADM1 ΔFERM-BM with vFLIP. The lysates were also subjected to immunoblotting to examine expression of vFLIP and Flag for wild-type and deletion mutants of CADM1. Error bars represent s.e.m. of triplicates.