The IKK or IRAK-1 assay was performed as described previously (20). In brief, IKKβ and IRAK-1 from the cytoplasmic extract (500 μg) was precipitated with respective Abs in the lysis buffer (20mM Tris-HCl (pH 7.6), 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 1% Triton X-100) followed by treatment with 50 μl of anti-rabbit IgG beads (eBioscience) and assayed in kinase assay buffer containing 20 mM HEPES (pH 7.6), 10 mM MgCl2, 1 mM DTT, 5 μCi [γ32P] ATP (NEN Life Science Products), 10 μM unlabeled ATP, and 0.5 μg of substrate (For IKKβ kinase: GST-IκBα (Santa Cruz Biotechnology); For IRAK-1 kinase: MBP (myelin basic protein) (Santa Cruz Biotechnology).
