Figure 3. Response to TLR3 and TLR4 ligands.

(A) SV40 fibroblasts were stimulated with increasing doses of poly(I:C) for 24 hours, and production of cytokines was assessed. C+ is a healthy control; UNC93B1^–/– served as a negative control. Values (mean ± SEM) were calculated from 3 independent experiments. (B) Native gel Western blot showing IRF3 dimers from total fibroblast cell lysates after stimulation with poly(I:C) for 1 or 2 hours. C+ and MYD88^–/– cells were used as positive controls to poly(I:C) stimulation, and UNC93B1^–/– was used as a negative control. (C) Nuclear protein extracts from SV40 fibroblasts stimulated for 30 minutes with IL-1β or TNF-α and 120 minutes with poly(I:C) were tested for the presence of the p65 subunit of NF-κB by ELISA. NEMO^–/– cells were used as a negative control for all stimuli; MyD88^–/– was used as a negative control for IL-1β; and UNC93B1^–/– was used for poly(I:C) stimulation. Values (mean ± SEM) were calculated from 3 independent experiments. NS, nonstimulated. (D) SV40 fibroblasts were stimulated with a TLR3-specific ligand, poly(A:U), transfected poly(I:C), RIG-I–specific ligand (7sk-as), or lipofectamine alone (L) for 24 hours and tested for cytokine production. Values (mean ± SEM) were calculated from 3 independent experiments. (E) PBMCs from healthy controls, various family members from kindred A (AII.1 and AII.3) and B (BII.3), and P1 and P2 were stimulated with LPS for 2 hours. Induction of IFNB1 and IL6 mRNA was assessed by real-time PCR. Values are expressed as relative fold change using the ΔΔCt method, where GUS was used for normalization; values (mean ± SEM) from 3 independent experiments were calculated.