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. 2007 May 14;204(5):1025–1036. doi: 10.1084/jem.20061825

Figure 1.

Figure 1.

Generation of IRAK4 kinase–inactive knock-in mice. (a) Domains of mouse IRAK4 protein. DD, death domain; UD, undetermined domain. K213/K214 were mutated to methionines in kinase-inactive knock-in mice. (b) Structure of the mouse IRAK4 gene, the targeting vector, and the targeted allele. Pr1 and Pr2 are primers used for genomic DNA PCR as described in c. (c) PCR analysis of IRAK4 genomic DNA in wild-type and IRAK4 kinase–inactive knock-in mice with Pr1 and Pr2. WT, wild-type; HET, heterozygote; KI, IRAK4 kinase–inactive knock-in mice. (d) In the top panel, lysates from mouse embryo fibroblasts from either WT or KI mice were left untreated or treated with IL-1β for 5 min. IRAK4 was immunoprecipitated from whole cell lysates using a rabbit polyclonal antibody to full-length IRAK4 and assayed for its ability to phosphorylate recombinant kinase domain of IRAK1 (residues 182–546) by immunoprecipitation kinase assay as described in Materials and methods. Recombinant HIS-tagged IRAK4 protein (rIRAK4) was included as a control. In the bottom panel, Western blot analysis depicting IRAK4 expression levels in whole cell lysates described in Materials and methods.