Poly 1 c tlr3

Bone marrow cells from wild-type mice were cultured in DMEM with 10% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin (Sigma), and 20% L929 supernatants to generate BMDMs. BMDMs were immortalized with J2 virus to generate iBMDMs. Cells were stimulated for the indicated times with LPS (Tlr4) (100 ng/ml), Pam3CSK4 (Tlr2/1) (100 nm), Pam2CSK4 (Tlr2/6) (100 ng/ml), and poly(I:C) (Tlr3) (25 μg/ml) (Sigma).

TLR agonists LPS from Escherichia coli serotype 026:B6 (TLR4), Pam3Cys (TLR2) and Poly(I:C) (TLR3) were purchased from Sigma (St. Louis, MO), Calbiochem (San Diego, CA), and Invivogen (San Diego, CA), respectively. Methylcarbamyl PAF C-16 (cPAF, a more metabolically stable analog) was purchased from Cayman Chemical (Ann Arbor, MI). NF-κB inhibitors acetyl-L-leucyl-L-leucyl-L-norleucinal (ALLN) and pyrrolidinedithiocarbamate (PDTC) were obtained from Tocris (Minneapolis, MN). CREB inhibitor KG-501 (2-naphthol-AS-E-phosphate) and PPARγ antagonist GW9662 were obtained from Sigma (St. Louis, MO). NF-κB p105/p50 inhibitor peptide was obtained from Novus Biologicals (Littleton, CO). Anti-COX2 Ab was supplied by Cayman (Ann Arbor, MI). Anti-iNOS, anti-phospho-IκBα (Ser32), anti-phospho-NF-κB p65 (Ser536), anti-phospho STAT3 (Tyr705), anti-β-actin, and secondary Abs goat anti-rabbit IgG and goat anti-mouse IgG conjugated to HRP were purchased from Cell Signaling Technology (Danvers, MA). Anti phospho-NF-κB p105/p50 (Ser337) was from SAB Signaling (College Park, MD).