|Detection of Mouse IL‑33 by Western Blot. Western blot shows lysates of mouse splenocytes. PVDF membrane was probed with 0.5 µg/mL of Rat Anti-Mouse IL‑33 Monoclonal Antibody (Catalog # MAB3626) followed by HRP-conjugated Anti-Rat IgG Secondary Antibody (Catalog # HAF005). Specific bands were detected for IL‑33 at approximately 18 and 35 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.|
|IL‑33 in bEnd.3 Mouse Cell Line. IL‑33 was detected in immersion fixed bEnd.3 mouse endothelioma cell line using Rat Anti-Mouse IL‑33 Monoclonal Antibody (Catalog # MAB3626) at 10 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Rat IgG Secondary Antibody (red, upper panel, Catalog # NL013) and counterstained with DAPI (blue, lower panel). View our protocol for Fluorescent ICC Staining of Cells on Coverslips.|
Intracellular Staining by Flow Cytometry
|Detection of IL‑33 in bEnd.3 Mouse Cell Line by Flow Cytometry. bEnd.3 mouse endothelioma cell line was stained with Rat Anti-Mouse IL‑33 Monoclonal Antibody (Catalog # MAB3626, filled histogram) or isotype control antibody (Catalog # MAB006, open histogram), followed by Phycoerythrin-conjugated Anti-Rat IgG Secondary Antibody (Catalog # F0105B). To facilitate intracellular staining, cells were fixed with Flow Cytometry Fixation Buffer (Catalog # FC004) and permeabilized with Flow Cytometry Permeabilization/Wash Buffer I (Catalog # FC005). View our protocol for Staining Intracellular Molecules.|
IL-33, also known as NF-HEV and DVS 27, is a 30 kDa proinflammatory protein that may also regulate gene transcription (1-3). DVS 27 was identifed as a gene that is upregulated in vasospastic cerebral arteries (1). NF-HEV was described as a nuclear factor that is preferentially expressed in the endothelial cells of high endothelial venules relative to endothelial cells from other tissues (2). IL-33 was identified based on sequence and structural homology with IL-1 family cytokines (3). DVS 27, NF-HEV, and IL-33 share 100% amino acid sequence identity. IL-33 is constitutively expressed in smooth muscle and airway epithelia. It is upregulated in arterial smooth muscle, dermal fibroblasts, and keratinocytes following IL-1 alpha or IL-1 beta stimulation (1, 3). Similar to IL-1, IL-33 can be cleaved in vitro by caspase-1, generating an N-terminal fragment that is slightly shorter than the C-terminal fragment (3, 4). The N-terminal portion of full length IL-33 contains a predicted bipartite nuclear localization sequence and a homeodomain-like helix-turn-helix DNA binding domain. By immunofluorescence, full length IL-33 localizes to the nucleus in HUVECs and transfectants (2). The C-terminal fragment, corresponding to mature IL-33, binds and triggers signaling through mast cell IL-1 R4/ST2L, a longtime orphan receptor involved in the augmentation of Th2 cell responses (3, 5-7). A ternary signaling complex is formed by the subsequent association of IL-33 and ST2L with IL-1R AcP (8). Stimulation of Th2 polarized lymphocytes with mature IL-33 in vitro induces IL-5 and IL-13 secretion (3). In vivo administration of mature IL-33 promotes increased production of IL-5, IL-13, IgE, and IgA, as well as splenomegaly and inflammatory infiltration of mucosal tissues (3). Full length and mature mouse IL-33 share approximately 55% and 90% amino acid (aa) sequence identity with human and rat IL-33, respectively. Mouse IL-33 shares less than 25% aa sequence identity with other IL-1 family proteins.
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Western Blot: Mouse IL‑33 Antibody [MAB3626]
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