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

<p>(A) Dual-luciferase assay using lysates of Hela cells expressing increasing amounts of CADM1 and vGPCR. HeLa cells were transfected with increasing amounts of either CADM1 or vGPCR with κB‐TATA Luc and pRL‐tk. After 36 hours, lysates were subjected to dual luciferase assays. The lysates were also subjected to immunoblotting to examine CADM1 and vGPCR expression using anti-Flag antibody. (B) HeLa cells expressing control scrambled shRNA or CADM1 shRNA were transfected with pRL-tk internal control Renilla luciferase plasmid, κB-TATA Luc and vGPCR as indicated. After 48 hours, lysates were subjected to dual luciferase assays. The lysates were also subjected to immunoblotting to examine vGPCR and CADM1 expression. (C) <i>Cadm1</i> ^<i>+/+</i>, <i>Cadm1</i> ^{<i>−/−</i>}, and <i>Cadm1</i> ^{<i>−/−</i>} MEFs reconstituted with wild-type Flag-CADM1 were transfected with vGPCR and 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 vGPCR expression using anti-Flag antibody. (D) 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 vGPCR. The lysates were also subjected to immunoblotting to examine expression of vGPCR and Flag for wild-type and deletion mutants of CADM1. Error bars represent s.e.m. of triplicates.