Figure 2. IL-1 induces the activation of IRAK1.

(A) Same as in Figure 1B, except that IRAK1 was immunoprecipitated with anti-IRAK1 or control IgG and the IPs were assayed for IRAK1 activity using GST-Pellino1 and Mg[γ³²P-ATP] as substrates in the presence (+) of IRAK4-IN-1 to inhibit co-immunoprecipitating IRAK4, and in the absence (−) or presence (+) of the IRAK1 inhibitor JNK-IN-7 (1 µM). (B) Same as in A, except that IRAK1 was assayed in the absence (−) or presence (+) of JNK-IN-7 (1 µM) or JNK-IN-8 (1 µM), and the membranes were additionally immunoblotted for all forms of IRAK1 — note that the unmodified form of IRAK1 disappears as it is gradually converted into more slowly migrating phosphorylated and ubiquitylated species after stimulation with IL-1β. (C) Same as in B, except that IRAK1 was immunoprecipitated from extracts of both IL-1R cells and IRAK1 KO IL-1R cells, and JNK-IN-7 was omitted from all assays. (D) Same as in B, except that IRAK4 was immunoprecipitated, and IRAK1 in the immunoprecipitates was assayed by including IRAK4-IN-1 in the assays.