anti human il 37  (R&D Systems)

Journal: Journal of Immunology Research

Article Title: Chloroquine and Rapamycin Augment Interleukin-37 Expression via the LC3, ERK, and AP-1 Axis in the Presence of Lipopolysaccharides

doi: 10.1155/2020/6457879

Figure Lengend Snippet: Effects of LPS and autophagy-modifying drugs on IL-37 and IL-1 β expression in U937 cells. (a) Relative fold changes of IL-37 and IL-1 β mRNA in U937 macrophages 2 h after treatments. Each sample was tested in triplicate, and the change folds were shown in Geomean ± S.E.M. (b) IL-37 and IL-1 β intracellular staining in U937 macrophages at 24 h after treatments. Brefeldin A was used to block protein transport. The experiment was repeated twice and yielded similar results. The representative graphs of flow cytometry analysis are shown accompanied with IL37-positive and IL-1 β -positive proportions in columns.

Article Snippet: After treatments with LPS and autophagy modifiers for 24 h and with Brefeldin A (eBioscience, San Diego, CA, USA) for the last 12 h, U937 cells and healthy human PBMCs were harvested and subjected to intracellular staining with primary antibodies including mouse anti-human IL-37 (clone 37D12, Invitrogen, San Diego, USA), anti-IL-1 β , and isotype IgG (BioLegend, USA), using the Fixation/Permeabilization Working Buffer (eBioscience) according to the manufacturer's manual.

Techniques: Expressing, Staining, Blocking Assay, Flow Cytometry

Journal: Journal of Immunology Research

Article Title: Chloroquine and Rapamycin Augment Interleukin-37 Expression via the LC3, ERK, and AP-1 Axis in the Presence of Lipopolysaccharides

doi: 10.1155/2020/6457879

Figure Lengend Snippet: Effects of LPS and autophagy-modifying drugs on IL-37 and IL-1 β expression in human PBMCs. (a) PBMCs from a healthy donor were isolated and treated with LPS and autophagy-modifying reagents for 24 h in vitro . Brefeldin A was also added to block protein transport for 12 h. Within the monocyte subgroups gated by their FS and SS characteristics, histograms of intracellular IL-37 staining detected by flow cytometry analysis are shown. (b) Columns indicate the proportions of intracellular IL-37-positive monocytes. (c) Columns indicate the proportions of intracellular IL-1 β -positive monocytes. (d) Supernatant IL-37 levels determined by ELISA, and each sample was assayed in triplicate (Mean ± S.D.).

Article Snippet: After treatments with LPS and autophagy modifiers for 24 h and with Brefeldin A (eBioscience, San Diego, CA, USA) for the last 12 h, U937 cells and healthy human PBMCs were harvested and subjected to intracellular staining with primary antibodies including mouse anti-human IL-37 (clone 37D12, Invitrogen, San Diego, USA), anti-IL-1 β , and isotype IgG (BioLegend, USA), using the Fixation/Permeabilization Working Buffer (eBioscience) according to the manufacturer's manual.

Techniques: Expressing, Isolation, In Vitro, Blocking Assay, Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Journal of Immunology Research

Article Title: Chloroquine and Rapamycin Augment Interleukin-37 Expression via the LC3, ERK, and AP-1 Axis in the Presence of Lipopolysaccharides

doi: 10.1155/2020/6457879

Figure Lengend Snippet: Effect of chloroquine administration on IL-37 expression, CD4 proliferation, and activation in rhesus monkeys. (a) Relative fold expression of IL-37 mRNA at 0, 3, and 7 days post chloroquine treatment in four rhesus monkeys. Each sample was assayed in triplicate and the change folds were shown in Geomean ± S.E.M. (b) Plasma IL-37 levels of monkeys were quantified by ELISA in triplicate (Mean ± S.D.). (c) Proportions of proliferating CD4+ T cells (Ki-67+CD4+CD3+) in peripheral blood at 0, 3, and 7 days post chloroquine treatment in monkeys. (d) Proportions of activated CD4+ T cells (CD38+HLA-DR+CD4+CD3+) in peripheral blood. (e) Spearman's correlation analysis between IL-37 mRNA and proportion of Ki-67+CD4. (f) Contents of IL-37, STAT3, and phosphorylated STAT3 in PBMCs of monkeys before and 3 and 7 days post chloroquine treatment. The experiment was repeated twice and yielded similar results. The representative western blot images are shown, and the ratio of IL-37 to housekeeping gene (actin) and the ratio of phosphorylated STAT3/STAT3 are shown in columns. Spearman's correlation analyses between (g) Ki-67+CD4 proportion or (h) IL-37 mRNA and phosphorylated STAT3 were also conducted and shown in graph.

Article Snippet: After treatments with LPS and autophagy modifiers for 24 h and with Brefeldin A (eBioscience, San Diego, CA, USA) for the last 12 h, U937 cells and healthy human PBMCs were harvested and subjected to intracellular staining with primary antibodies including mouse anti-human IL-37 (clone 37D12, Invitrogen, San Diego, USA), anti-IL-1 β , and isotype IgG (BioLegend, USA), using the Fixation/Permeabilization Working Buffer (eBioscience) according to the manufacturer's manual.

Techniques: Expressing, Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Journal of Immunology Research

Article Title: Chloroquine and Rapamycin Augment Interleukin-37 Expression via the LC3, ERK, and AP-1 Axis in the Presence of Lipopolysaccharides

doi: 10.1155/2020/6457879

Figure Lengend Snippet: IL-37 expression induced by LPS and autophagy-modifying reagents correlates with LC3 conversion. (a) The representative western blot images and (b) columns of relative densitometric data showing the content of Beclin1, p62, and LC3 in treated U937 cells. The experiment was repeated twice independently and yielded similar results. (c) Spearman's analysis revealed a correlation between IL-37 mRNA and the LC3 II/I ratio only in the presence of LPS stimulation. (d) The representative images of LC3 immunofluorescence staining observed with a confocal microscope. (e) Comparison of IL-37 expression and IL-1 β expression between Cyto-ID-positive cells and Cyto-ID-negative cells isolated by flow cytometry sorting (quantitation was repeated three times and presented as Mean ± S.D.). The ratio of IL-37 and IL-1 β expression in Cyto-ID-positive cells to negative cells was also computed and plotted in the right.

Article Snippet: After treatments with LPS and autophagy modifiers for 24 h and with Brefeldin A (eBioscience, San Diego, CA, USA) for the last 12 h, U937 cells and healthy human PBMCs were harvested and subjected to intracellular staining with primary antibodies including mouse anti-human IL-37 (clone 37D12, Invitrogen, San Diego, USA), anti-IL-1 β , and isotype IgG (BioLegend, USA), using the Fixation/Permeabilization Working Buffer (eBioscience) according to the manufacturer's manual.

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Microscopy, Isolation, Flow Cytometry, Quantitation Assay

Journal: Journal of Immunology Research

Article Title: Chloroquine and Rapamycin Augment Interleukin-37 Expression via the LC3, ERK, and AP-1 Axis in the Presence of Lipopolysaccharides

doi: 10.1155/2020/6457879

Figure Lengend Snippet: Cell signaling pathways contributing to the inductive transcription of IL-37. (a) The effect of LPS and autophagy-modifying reagents on the contents of phosphorylated NF- κ B p65/I κ B α and phosphorylated AP-1 (c-Fos/c-Jun) proteins. Two independent experiments were repeated and yielded similar results. The representative western blot images were shown. (b) The effect of MAPK inhibitors and NF- κ B inhibitor on the expression of IL-37 and IL-1 β induced by LPS and rapamycin treatment in U937 cells. Each sample was assayed in triplicate, and the results were shown in Geomean ± S.E.M. Inhibitors used in this study included SB 203580 (100 μ M), U0126 (200 μ M), SP600125 (20 μ M), and BAY 11-7082 (10 μ M). S.S.U.B: combination of the four inhibitors. (c) The effect of MAPK agonists on IL-37, IL-1 β , and IL-18 expression in untreated U937 macrophages was also investigated. The agonists were LM22B-10 at 125, 250, 500, and 1000 nM and anisomycin at 1.25, 2.5, 5, and 10 μ M. Each sample was assayed in triplicate (Geomean ± S.E.M.). (d) The effect of LPS and autophagy-modifying reagents on the contents of phosphorylated Erk1/2 and phosphorylated p38 MAPK. Two independent experiments were repeated and yielded similar results. The representative western blot images were shown.

Article Snippet: After treatments with LPS and autophagy modifiers for 24 h and with Brefeldin A (eBioscience, San Diego, CA, USA) for the last 12 h, U937 cells and healthy human PBMCs were harvested and subjected to intracellular staining with primary antibodies including mouse anti-human IL-37 (clone 37D12, Invitrogen, San Diego, USA), anti-IL-1 β , and isotype IgG (BioLegend, USA), using the Fixation/Permeabilization Working Buffer (eBioscience) according to the manufacturer's manual.

Techniques: Western Blot, Expressing