Figure 3.

Reduced NF-κB activation by IL-18 in IRAK-deficient Th1 cells. (A) Reduced degradation of IκB-α in IRAK-deficient cells. IRAK-deficient [IRAK(−)] and wild-type cells were stimulated with IL-18 (50 ng/ml) for different time courses as indicated. Cell lysates were prepared and separated on SDS-PAGE. IκB-α was detected by Western blot analysis using an IκB-α–specific antibody. Amounts of protein loading were determined by probing the same filter with antibody specific for ERK-2. (B) Reduced NF-κB activation by IL-18 in IRAK-deficient cells. Cells were stimulated with IL-18 (50 ng/ml), TNF-α (50 ng/ml), or PMA (50 ng/ml) plus ionomycin (0.125 μM) for 30 min. Nuclear extracts were prepared and incubated with ³²P-labeled NF-κB specific double-stranded oligonucleotide. The NF-κB DNA complex was separated on 6% polyacrylamide gel in 0.5% Tris-borate-EDTA.