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. 2017 Oct 16;199(10):3654–3667. doi: 10.4049/jimmunol.1700443

FIGURE 4.

FIGURE 4.

Effect of IRAK1 mutation at Lys134 on CpG-induced K48-linked ubiquitination and degradation of IRAK1. (A) IRAK1−/− IMM were transfected with different amounts of WT or Lys134 mutant IRAK1 (K134R) plasmid DNA for 48 h. WCL were analyzed by immunoblotting with anti-IRAK1 and anti–β-actin Abs. (B) IRAK1−/− IMM were transfected with WT or K134R IRAK1 DNA, followed by treatment with CpG for the indicated periods of time. WCL were immunoprecipitated with anti-IRAK1 Ab, followed by immunoblotting with anti-ubiquitin K48-linkage specific and anti–β-actin Abs. (C) IRAK1−/− IMM were transfected with WT or K134R IRAK1 DNA, followed by exposure to CpG with or without Dex for the indicated periods of time. Dex was cotreated with the TLR ligands. WCL were analyzed by immunoblotting with anti-IRAK1 and anti–β-actin Abs. Western blots were quantified by densitometric analyses. (A and B) The abundance of IRAK1 was normalized to β-actin. IRAK1 abundance in untreated, resting IRAK1+/+ IMM was considered as 1 U. (C) The abundance of K48 linkage–specific IRAK1 ubiquitination was normalized to β-actin. IRAK1 ubiquitination in untreated, resting cells was considered as 1 U. The densitometry data presented are mean ± SD. Western blots are from a single experiment that are representative of three independent experiments. *p < 0.05 versus IRAK1−/− IMM treated with WT IRAK1 DNA and CpG.