Fig. 5.

IL-1R–dependent signaling in the patient’s fibroblasts. (A) IκB-α protein degradation in SV40-fibroblast lines from a healthy control, the IRAK-1–deficient patient, and IRAK-4– and MyD88-deficient patients, left unstimulated or stimulated with IL-1β (10 ng/mL) or TNF-α (20 ng/mL) for various periods of time (min), analyzed by Western blotting. Similar results were obtained in three independent experiments. (B) NF-κB translocation, assessed by EMSA, in SV40-fibroblasts from a healthy control, the IRAK-1–deficient patient, and IRAK-4– and MyD88-deficient patients, following stimulation with IL-1β (10 ng/mL) and TNF-α (20 ng/mL) for 20 min. Similar results were obtained in three independent experiments. (C) NF-κB–dependent transcription was assessed with the NF-κB reporter luciferase assay in SV40-fibroblasts derived from a healthy control, the IRAK-1–deficient patient, and IRAK-4–, MyD88-, and MECP2-deficient patients. Cells were transiently transfected with Renilla luciferase- and NF-κB luciferase-encoding vectors; 24 h after transfection, the cells were left untreated or were stimulated with IL-1β (10 ng/mL) or TNF-α (20 ng/mL) for 42 h and then harvested. Reporter gene activities were measured and the values obtained were normalized for transfection efficiency on the basis of Renilla luciferase expression. The values shown (means ± SEM) were obtained in three independent experiments. (D) IL-8 secretion by SV40-fibroblasts from healthy controls (n = 3), the IRAK-1–deficient patient, and IRAK-4–, MyD88-, and MECP2-deficient patients, left unstimulated or stimulated with IL-1β (10 ng/mL), polyIC (25 μg/mL), and TNF-α (20 ng/mL) as a positive control. The values shown (means ± SEM) were obtained in three independent experiments. *P < 0.05.