|Detection of Mouse IL‑33 by Western Blot. Western blot shows lysates of HEK293T human embryonic kidney cell line either mock transfected or transfected with full length mouse IL-33. PVDF membrane was probed with 0.4 µg/mL of Goat Anti-Mouse IL‑33 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3626) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF109). A specific band was detected for IL‑33 at approximately 37 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.|
|IL‑33 in Mouse Spleen. IL‑33 was detected in immersion fixed frozen sections of mouse spleen using Goat Anti-Mouse IL‑33 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3626) at 5 µg/mL overnight at 4 °C. Tissue was stained using the Anti-Goat HRP-DAB Cell & Tissue Staining Kit (brown; Catalog # CTS008) and counterstained with hematoxylin (blue). Specific staining was localized to cytoplasm in lymphocytes. View our protocol for Chromogenic IHC Staining of Frozen Tissue Sections.|
Intracellular Staining by Flow Cytometry
|Detection of IL‑33 in Mouse Splenocytes by Flow Cytometry. Mouse splenocytes were treated for 5 hours with 50 ng/mL PMA and 1 μg/mL Ca2+ ionomycin then stained with Goat Anti-Mouse IL‑33 Antigen Affinity‑purified Polyclonal Antibody (Catalog # AF3626, filled histogram) or isotype control antibody (Catalog # AB-108-C, open histogram), followed by Phycoerythrin-conjugated Anti-Goat IgG Secondary Antibody (Catalog # F0107). To facilitate intracellular staining, cells were fixed with paraformaldehyde and permeabilized with saponin.|
IL‑33 in bEnd.3 Mouse Cell Line. IL‑33 was detected in immersion fixed bEnd.3 mouse endothelioma cell line using Goat Anti-Mouse|
IL‑33 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3626) at 10 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Goat IgG Secondary Antibody (red, upper panel; Catalog # NL001) and counterstained with DAPI (blue, lower panel). View our protocol for Fluorescent ICC Staining of Cells on Coverslips.
Proliferation Induced by IL‑33 and Neutralization by Human IL‑33 Antibody. Recombinant Mouse IL‑33 (Catalog #|
3626-ML) stimulates proliferation in the D10.G4.1 mouse helper T cell line in a dose-dependent manner (orange line), as measured by Resazurin (Catalog # AR002). Proliferation elicited by Recombinant Mouse IL‑33 (0.25 ng/mL) is neutralized (green line) by increasing concentrations of Goat Anti-Mouse IL‑33 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3626). The ND50 is typically 10-50 ng/mL.
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% 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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