Intracellular Staining by Flow Cytometry
|Detection of IL‑18/IL‑1F4 in Human PBMCs by Flow Cytometry. Human peripheral blood mononuclear cells (PBMCs) either untreated (upper panel) or treated with LPS for 24 hours (lower panel) were stained with Mouse Anti-Human IL‑18/IL‑1F4 Propeptide Monoclonal Antibody (Catalog # MAB646, filled histogram) or isotype control antibody (Catalog # MAB002, open histogram), followed by Phycoerythrin-conjugated Anti-Mouse IgG Secondary Antibody (Catalog # F0102B). To facilitate intracellular staining, cells were fixed with paraformaldehyde and permeabilized with saponin.|
Pro-IL-18 (pro-Interleukin 18; also pro-IGIF and pro-IL-1 gamma ) is a 24 kDa member of the IL-1 family of molecules. It is widely expressed, being produced by keratinocytes, intestinal epithelium, T cells, macrophages and osteoblasts. Human Pro-IL-18 is 193 amino acids (aa) in length. Although mature IL-18 induces IFN-gamma secretion by NK and T cells, Pro-IL-18 appears to have little intrinsic activity. Generally, active IL-18 is considered to arise from caspase-1 cleavage of Pro-IL-18 between Asp36-Tyr37. This generates an 18 kDa mature C-terminal fragment, and a 4 kDa (predicted) N-terminal prosegment that runs at 6 kDa in SDS-PAGE. Other proteases are known to process Pro-IL-18. Caspase-3 cleavage after Asp68 generates an inactive 14 kDa mature segment, Merpin beta -subunit cleavage after Asn52 generates a marginally active 17 kDa mature segment, while parasite Cys protease cleavage after Val47 generates an inactive 17 kDa mature molecule. One splice variant shows a deletion of aa 27-30. Over aa 2-36, human Pro-IL-18 shares 63% aa identity with mouse Pro-IL-18.
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