The reporter gene plasmids were as described EPZ-6438 previously 34. The IRF7-Flag plasmid was a generous gift from Professor Paul Moynagh (NUIM). The IRF3 and IRF7-YFP plasmids were a generous gift from Professor Taniguchi (University of Tokyo). The RIG-I and Mda-5 mammalian expression plasmids were gifts from Professor Steve Goodbourn, University of London. Mal/TIRAP−/−

and TRIF−/− mice were constructed as described previously 5, 17. Mal/TIRAP KO and TRIF−/− mice were on a C57BL/6 background. All mice were confirmed as being homozygous mutants by PCR genotyping of DNA. All the animal protocols used in this study were approved by the Ethical Committee at the National University of Ireland, Maynooth and in accordance with the Animals (Scientific Procedures) Act, 1986, UK. BM-derived macrophages (BMDM) were generated by differentiation

of age- and sex-matched C57BL/6, Mal−/− and TRIF−/− mice for 8 days in complete DMEM medium supplemented with L929-conditioned supernatants. Immortalised cell lines from WT, Mal−/− and TRIF−/− mice were established by infecting primary BM cells with the J2 recombinant retrovirus as described previously 6, 35, 36. Cell lines showed similar patterns of surface receptor expression, see more activation markers and cytokine production in response to various TLR ligands when compared with primary BMDM. Total RNA was isolated from all types of cells using the TRIzol® Reagent according to the manufacturer’s instructions (Invitrogen). Thereafter, total RNA was converted to first strand cDNA as described previously 37. Total cDNA was used as starting material for real-time RT-PCR quantitation with DyNAmo®HS SYBR Green kit (Finnzymes) on a real-time PCR system (DNA Engine OPTICON® system; MJ Research). For the amplification of the specific genes, the Protein kinase N1 following primers were used; mIFN-β,

forward, GGAGATGACGGAGAAGATGC, and reverse, CCCAGTGCTGGAGAAATTGT; hIFN-β, forward, AACTGCAACCTTTCGAAGCC, and reverse, TGTCGCCTACTACCTGTTGTGC; mTNFα, forward, CATCTTCTCAAAATTCGAGTGACAA, and reverse, TGGGAGTAGACAAGGTACAACCC; hTNFα, forward, CACCACTTCGAAACCTGGGA, and reverse, CACTTCACTGTGCAGGCCAC; mMal/TIRAP, forward, GCTTCATCCTCCTCCGT, and reverse, TGTGTTGGTGGCGAGGT; mTLR3, forward, GTGAGTCTGAAGTACCTAAGTC, and reverse, GAACTGGTAGACAGTTGGAGGT. For each mRNA quantification, the housekeeping gene hypoxanthine phosphoribosyltransferase 1 (HPRT) was used as a reference point using the following primers; mHPRT forward, CCCTGAAGTACTCATTATAGTCAAGGGCAT, and reverse, GCTTGCTGGTGAAAAGGACCTCTCGAAG; hHPRT forward, AGCTTGCTGGTGAAAAGGAC, and reverse, TTATAGTCAAGGGCATATCC. Real-time PCR data were analyzed using 2−ΔΔCT method as described previously 38. Human Mal lentiviral shRNA plasmids were from Sigma-Aldrich (Mal MISSION® shRNA). THP1 cells were lentivirally transduced with either the control plasmid (pLKO.1-puro, SCH001) or the plasmid-encoding shRNA specific for Mal/TIRAP (Tirap MISSION® shRNA NM_052887, TRC No. TRCN0000005565).