FIG. 5.
IRAK1c inhibits IL-1β-induced NF-κB activation. (A) 293 cells were transfected with 250 ng of the indicated myc-IRAK1 and FLAG-MyD88 plasmids and 1 μg of pHTS-NF-κB-luciferase plasmid. Cells were nonstimulated (−) or stimulated (+) with IL-1β (5 ng/ml) for 12 h, and cell extracts were analyzed for luciferase activity in replicates of four. The means and standard deviations are plotted. (B) G292 cells were transfected with the indicated myc-IRAK1 constructs and 2 μg of pHTS-NF-κB-luciferase plasmid. Cells were nonstimulated (−) or stimulated (+) with IL-1β (5 ng/ml) for 12 h, and cell extracts were analyzed for luciferase activity in replicates of five. (C) 293 cells (solid line) or G292 cells (dashed line) were transfected with increasing amounts of myc-IRAK1c, and NF-κB-luciferase activity was analyzed as described above. Percent activation compared to vector-transfected cells was calculated and plotted. (D) G292 cells were transfected with 250 ng of myc-IRAK1c and the indicated amounts of FLAG-TRAF6 plasmid. Cells were stimulated and analyzed as described above. (E) IRAK1−/− fibroblasts reconstituted with IRAK1, IRAK1-KD, and IRAK1c were stimulated with 5 ng/ml of IL-1β for 30 min. Whole-cell lysates were prepared directly in sample buffer and subjected to immunoblot analysis with anti-IκBα (top panel), p-cJUN (middle panel), and anti-actin antibodies (bottom panel).