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

FIGURE 5.

FIGURE 5.

GC targets IRAK1 ubiquitination in suppressing TLR ligand–induced inflammatory actions. IRAK1−/− IMM were transfected with WT or K134R IRAK1 DNA, followed by exposure to (A) CpG and (B) LPS, with or without Dex for 30 min. Dex was cotreated with the TLR ligands. 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 30 min. Dex was cotreated with the TLR ligands. WCL were analyzed by Western blot using anti–phospho-TAK1 (p-TAK1), total TAK1; anti–phospho-IκBα (p-IκBα), total IκBα; anti–phospho-JNK (p-JNK), total JNK; anti–phospho-p38 MAPK (p-p38) and total p38 MAPK (p38) Abs. All Western blots were quantified by densitometric analyses. (A and B) The abundance of K48 linkage–specific IRAK1 ubiquitination was normalized to β-actin. IRAK1 ubiquitination in untreated, resting cells was considered as 1 U. (C) Abundance of phosphorylated proteins was normalized to total protein or β-actin, as appropriate. Protein expression in untreated, resting cells was considered as 1 U. The densitometry data presented are mean ± SD. *p < 0.05 versus IRAK1−/− IMM treated with WT IRAK1 DNA and CpG. Western blots are from a single experiment and are representative of three independent experiments. (D and E) IRAK1+/+ or IRAK1−/− IMM were transfected with WT or K134R IRAK1 DNA, followed by the treatment with (D) CpG or (E) LPS in the presence or absence of Dex for another 18 h. Concentrations of IL-6, TNF-α, and IL-12 in the culture media were analyzed by ELISA. Data presented as mean ± SEM from three to four independent experiments. *p < 0.05 versus IRAK1−/− IMM treated with WT IRAK1 DNA and CpG, #p < 0.05 versus IRAK1−/− IMM treated with WT IRAK1 DNA and (CpG plus Dex). ns, not significant.