Mouse IL-18 BPc Antibody

Detection of Mouse IL-18 BPc by Western Blot LPS-induced IL-18 levels are regulated by LXR activation.(a) BMDM were treated with vehicle (DMSO), GW3965 (1 μ mol/L) or LXR antagonist (LXR ant, 1 μ mol/L) for 24 hours with or without LPS (100 ng/ml) for the last 6 hours. IL-18 mRNA levels were analyzed by RT-qPCR. Values indicate expression normalized to cyclophilin and are presented relative to the expression in LPS-treated cells, set as 1. Values represent the mean ± SEM (3–4 different BMDM preparations). t-test: ***p ≤ 0.001, ns p > 0.05. (b) IL-18 mRNA expression in BMDM from WT and LXR alpha beta −/− mice were analyzed by RT-qPCR. Shown is a representative experiment performed in triplicate (mean ± SD) t-test: *p ≤ 0.05, ns p > 0.05. (c) BMDM were treated as in A and then activated with ATP for the last 2 hours. Pro-IL-18 expression was analyzed by immunoblotting and Hsp90 levels were assayed as loading control. Shown is a representative experiment of three. (d) BMDM were treated as in A. Intracellular expression of IL-18 is shown as analyzed by immunoblotting. Samples shown were analysed on non consecutive lanes of the same blot. (e) BMDM were treated as indicated. Secreted IL-18 levels in culture supernatants were quantified by ELISA. Values represent mean ± SEM (n = 3). t-test: *p ≤ 0.05. Image collected and cropped by CiteAb from the following open publication (https://www.nature.com/articles/srep25481), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse IL-18 BPc by Western Blot Identification of IL-18BP as a novel LXR target gene.(a) BMDM were treated with vehicle (DMSO) or GW3965 (GW) for 24 hours and IL-18BP mRNA content was analyzed by RT-qPCR. Values indicate expression normalized to cyclophilin and are presented relative to the expression in vehicle-treated cells. Values represent the mean of 4 independent experiments ± SEM. t-test: ***p ≤ 0.001. (b) BMDM from WT or LXR alpha beta deficient mice (LXR alpha beta −/−) were treated as in A with or without the RXR activator LG268 (LG). IL-18BP mRNA content was analyzed as in A. Values represent the mean of 3 independent experiments ± SEM. *p ≤ 0.05, **p ≤ 0.01. (c) BMDM were treated with vehicle (DMSO) or GW3965 (GW) for 24 hours supplemented with Brefeldin A for the last 6 hours. Protein content was analyzed by immunoblotting and Hsp90 levels were assayed as loading control. (d) RAW264.7 cells were transfected with hLXR alpha or pcDNA3.1 (− ) along with the indicated IL-18BP luciferase reporters or pGL3 empty vector. Cells were treated as described in the Methods section. For each reporter, luciferase and beta -galactosidase activities were measured and the ratio was compared to the vehicle-treated condition in the absence of LXR alpha (− ), which was set as 1. Data are mean value ± SD (n = 3) of one representative experiment. t-test: **p ≤ 0.01, ***p ≤ 0.001. (e) Upper panel, location of a putative LXRE in the IL-18BP locus. Arrows indicate the position of primers used for ChIP assays. Lower panel, BMDM cells were incubated with or without GW3965 at 1 μ mol/L for 2 hours. LXR occupancy was assessed by ChIP assays. Primers shown in upper panel were used to amplify an unrelated site at − 4.7 kb that served as a negative control, the − 1.1 kb putative LXRE, − 0.5 kb and TSS as GW3965 responsive sites. SREBP1c primers were used as a positive control for LXR binding. Shown is a representative experiment of three. Image collected and cropped by CiteAb from the following open publication (https://www.nature.com/articles/srep25481), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse IL-18 BPc by Western Blot Identification of IL-18BP as a novel LXR target gene.(a) BMDM were treated with vehicle (DMSO) or GW3965 (GW) for 24 hours and IL-18BP mRNA content was analyzed by RT-qPCR. Values indicate expression normalized to cyclophilin and are presented relative to the expression in vehicle-treated cells. Values represent the mean of 4 independent experiments ±  SEM. t-test: ***p ≤  0.001. (b) BMDM from WT or LXR alpha beta deficient mice (LXR alpha beta −/−) were treated as in A with or without the RXR activator LG268 (LG). IL-18BP mRNA content was analyzed as in A. Values represent the mean of 3 independent experiments ±  SEM. *p ≤  0.05, **p ≤  0.01. (c) BMDM were treated with vehicle (DMSO) or GW3965 (GW) for 24 hours supplemented with Brefeldin A for the last 6 hours. Protein content was analyzed by immunoblotting and Hsp90 levels were assayed as loading control. (d) RAW264.7 cells were transfected with hLXR alpha or pcDNA3.1 (− ) along with the indicated IL-18BP luciferase reporters or pGL3 empty vector. Cells were treated as described in the Methods section. For each reporter, luciferase and beta -galactosidase activities were measured and the ratio was compared to the vehicle-treated condition in the absence of LXR alpha (− ), which was set as 1. Data are mean value ±  SD (n =  3) of one representative experiment. t-test: **p ≤  0.01, ***p ≤  0.001. (e) Upper panel, location of a putative LXRE in the IL-18BP locus. Arrows indicate the position of primers used for ChIP assays. Lower panel, BMDM cells were incubated with or without GW3965 at 1 μ mol/L for 2 hours. LXR occupancy was assessed by ChIP assays. Primers shown in upper panel were used to amplify an unrelated site at − 4.7 kb that served as a negative control, the − 1.1 kb putative LXRE, − 0.5 kb and TSS as GW3965 responsive sites. SREBP1c primers were used as a positive control for LXR binding. Shown is a representative experiment of three. Image collected and cropped by CiteAb from the following open publication (https://www.nature.com/articles/srep25481), licensed under a CC-BY license. Not internally tested by R&D Systems.