Mammalian expression vectors for human Flag- or HA-tagged RIG-I, RIG-I-N (containing only the N-terminal CARD domain, 1 to 284 amino acids), MAVS, TBK1, IKKε, IRF3 and its mutant IRF3-5D (the phosphorylation sites as Ser396, Ser398, Ser402, Thr404, and Ser405 residues were replaced by aspartic acid in the C-terminal end of IRF-3) (47 (link)), ubiquitin and its mutants K48 and K63 ubiquitin, IFN-β promoter luciferase reporter, ISRE, and NF-κB luciferase reporter constructs were described previously (10 (link), 39 (link), 48 (link)). The full-length human MAP4K1 was cloned into mammalian expression vector pRK5 with an N-terminal HA or Flag tag and pcDNA3.1 with an N-terminal Myc tag by standard molecular biology techniques. MAP4K1-specific siRNA constructs were generated by the pSuper.retro vector (OligoEngine, Seattle, WA, USA) with double-strand oligonucleotides targeting MAP4K1 sequences using protocols recommended by the manufacturer. The following sequences were designed for targeting human MAP4K1 mRNA: MAP4K1-RNAi 1, 5′-CCTGGATCTTCTTGACAAA -3′; MAP4K1 RNAi 2, 5′-CCTGATCCTGGATCTTCTT -3′, and MAP4K1 RNAi 3, 5′-CCAGACGCCTCTCTTTCAT -3′. The following shRNA sequences were used: control shRNA sequence (5′-CAACAAGATGAAGAGCACCAA -3′) and Dtx4 shRNA (5′-TTAAGGCAGCCGTGGTCAATG -3′).
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