Fig 2. L pro cleaves MAVS and TBK1. (A) HeLa R19 cells were infected at MOI 10 with the indicated viruses and lysed at 2,4,6,8 h pi. Cell lysates were subjected to Western Blot analysis for MAVS, TBK1, IRF3, eIF4GI, G3BP1, EMCV capsid proteins and tubulin. (B) HEK293T cells were transfected with 1 μ g pcDNA-FLAG-TBK1 plasmid and 0.5 μ g pIRES-GFP-L pro plasmid. 16 h post transfection cells were lysed and lysates were subjected to Western Blot analysis for FLAG and GFP. (C) His-hTBK1 was incubated with 0–3 μ g sL pro or 3 μ g sL pro C51A for 2 h and reaction mixtures were subsequently subjected to Western Blot analysis for His. (D) HeLa OHIO cells were transfected with 7 μ g pCS2- 6Myc-mTBK1 or pCS2-6Myc-mTBK1 692AAA694 . 24 h post transfection, cells were lysed and incubated with 2 μ g recombinant sL pro for 2 h before being subjected to Western Blot analysis for HA. (E) Comparison of TBK1 amino acids 661–729 of human and mouse origin. Asterisks denote conserved residues. KLK at position 692–694 was mutated to AAA.