Puno mtlr9 ha

TLR9 murine agonist CpG (ODN1826), TLR9 murine antagonist CpG (ODN2088), monoclonal anti-mTLR9 antibody, mouse monoclonal anti-hemagglutinin-tag (HA) Ab, pUNO-mTLR9-HA, pUNO-mcs purchased from Invivogen (San Diego, CA). Avian Myeloblastosis Virus-Reverse Transcriptase (AMV-RT) enzyme, SYBR Green Jump Start ready mix, Lysostaphin, actinomycin-D, and Tricarbonyl-dichloro-ruthenium (II) dimer (CORM2) were obtained from Sigma Aldrich (St. Louis, MO). The Micro-Bicinchoninic Acid assay (BCA) protein assay kit was purchased from Pierce Biotechnology (Rockford, IL). The Cobalt Protoporphyrin-IX (CoPP), Zinc Protoporphyrin-IX (ZnPP), Biliverdin was from Frontier Scientific, Logan, UT. The RNA isolation kit was acquired from Qiagen (Valencia, CA). Murine PMCs were isolated from HO-1^+/+ and HO-1^-/- mice and cultures were established as reported earlier [6 (link)].

Expression plasmids containing a human (h)TLR9 gene (pUNO-hTLR9^HA) and mouse (m)TLR9 (pUNO-mTLR9^HA) were obtained from InvivoGen, and TLR9^YFP (pcDNA3-TLR9-YFP) was obtained from Addgene. The plasmid phRL-TK constitutively expressing Renilla luciferase for the normalization of transfection efficiency was obtained from Promega. Plasmid coding for firefly luciferase under the NF-κB promoter (pELAM-1-luciferase) was a gift from C. Kirschning (Institute for Medical Microbiology, University of Duisburg-Essen, Essen, Germany). For site-directed mutagenesis, C-terminal HA-tagged hTLR9 (hTLR9^HA) was used. The chimeras between hTLR9 and mTLR9, inserted into the pUNO vector, are depicted in Supplementary Table S1. Cells were treated with different TLR9 agonists (Supplementary Table S2 depicts nucleotide sequences): human-specific ODN2006 (h2006) and minH75 (H75)^{16 (link)} and mouse-specific ODN1826 (m1826) and minM80 (M80)^{15 (link)} (all B-class ODNs), with a PTO backbone, a PD backbone, or a mixed PTO–PD backbone (Integrated DNA Technologies). The 3′- and 5′-end FITC labeled M80 were obtained from Integrated DNA Technologies.