Figure 2. Regulation of IL-18BP expression is dependent on IRF8.
(a) BMDM were transfected with control (Ctrl) or IRF8 siRNAs for 24 hours and treated vehicle (DMSO) or GW3965 (1 μ mol/L) for 24 hours. IL-18BP mRNA content was analyzed by RT-qPCR. Values indicate expression normalized to cyclophilin and are presented relative to the expression in siRNA-control vehicle-treated cells, which was set as 1. A representative experiment is shown (mean ± SD) t-test: ***p ≤ 0.001. (b) BMDM from WT or IRF8−/− mice were cultured with T1317 (T) (1 μ mol/L) for 24 hours. IL-18BP mRNA content was analyzed by RT-qPCR. Values indicate expression normalized to cyclophilin and are presented relative to the expression in siRNA-control vehicle-treated cells, which is set as 1. A representative experiment is shown (mean ± SD) t-test: **p ≤ 0.01. (c) BMDM were incubated as in Fig. 1E. IRF8 occupancy was determined by ChIP assays. Primers shown in Fig. 1E were used to amplify the − 0.5 kb and TSS putative IRF8 binding sites. Primers amplifying Cystatin C (CystC) and β -actin (β -act) were used as positive and negative controls, respectively. Background occupancy using IgG in the absence or presence of ligand is depicted by light and dark grey bars respectively. Shown is a representative experiment. (d) RAW-LXRα cells were treated with vehicle (DMSO) or GW3965 (GW) (1 μ mol/L) for 24 h. IRF8 was immunoprecipitated and tyrosine phosphorylation of IRF8 was analysed by immunoblotting. Protein content in the cell lysate prior to immunoprecipitation (Input) was analysed. A representative experiment is shown.