Myd88 / Trif / mice were transfected with 3.3 µg/ml c-di-GMP or pdA:dT (DNA), or overlayed with 100 ng/ml LPS as indicated. After 6 h of stimulation, induction of IFN- mRNA was analyzed by quantitative RT-PCR, with normalization to ribosomal protein rps17 message. n.d., not detectable. (B) As in A, except Tnfr1 / or Tnfr1 / Tbk1 / macrophages were analyzed. Tbk1 deficiency is embryonic lethal, but viability can be rescued on the TNF receptor 1 (Tnfr1)-deficient background. (C) As in A except Ikbke (IKK/IKK-i)-deficient mice were analyzed. (D) as in A, except Irf3 / and Irf7 / macrophages were analyzed. (E) as in A, except macrophages deficient in the type I IFN receptor (Ifnar / ) were analyzed. (F) as in A, except Irf5 / and Irf1 / macrophages were analyzed. Results are representative of at least three independent experiments. Data are means ± SD (n = 3). *, P < 0.05; and **, P < 0.001 as compared with wild-type bone marrow macrophages (Student's t test) . discrepancy between our results and those of Karaolis et al. (2007a) , it is more likely that we were able to detect MAP kinase activation because transfection of c-di-GMP triggers more robust responses than overlayed c-di-GMP, and because we examined later time points.