Detection of Human OX40/TNFRSF4 by Western Blot.
Western blot shows lysates of human T regulatory cells (Tregs) and human peripheral blood mononuclear cells (PBMCs) untreated (-) or treated (+) with 1 μg/mL PHA for 5 days. PVDF membrane was probed with 0.5 µg/mL of Sheep Anti-Human OX40/TNFRSF4 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3388) followed by HRP-conjugated Anti-Sheep IgG Secondary Antibody (Catalog # HAF016). A specific band was detected for OX40/TNFRSF4 at approximately 45 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.
Detection of OX40/TNFRSF4 in Human T Cells by Flow Cytometry.
Human T cells were treated for 3‑5 days with 1 μg/mL PHA then stained with Sheep Anti-Human OX40/ TNFRSF4 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3388, filled histogram) or control antibody (Catalog # 5-001-A, open histogram), followed by NorthernLights™ 557-conjugated Anti-Sheep IgG Secondary Antibody (Catalog # NL010).
OX40/TNFRSF4 in Human Tonsil.
OX40/TNFRSF4 was detected in immersion fixed paraffin-embedded sections of human tonsil using Sheep Anti-Human OX40/TNFRSF4 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF3388) at 3 µg/mL for 1 hour at room temperature followed by incubation with the Anti-Sheep IgG VisUCyte™ HRP Polymer Antibody (Catalog # VC006). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to plasma membrane. View our protocol for IHC Staining with VisUCyte HRP Polymer Detection Reagents.
Preparation and Storage
Reconstitute at 0.2 mg/mL in sterile PBS.
Reconstitution Buffer Available
The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below. *Small pack size (SP) is shipped with polar packs. Upon receipt, store it immediately at -20 to -70 °C
Stability & Storage
Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
12 months from date of receipt, -20 to -70 °C as supplied.
1 month, 2 to 8 °C under sterile conditions after reconstitution.
6 months, -20 to -70 °C under sterile conditions after reconstitution.
OX40 (CD134; TNFRSF4) is a T cell costimulatory molecule of the TNF receptor superfamily that coordinates with other membrane-bound costimulators such as CD28, CD40, CD30, CD27 and 4-1BB (1‑3). OX40 is expressed on naïve CD4+ T cells only after engagement of the TCR by antigen presenting cells (APC; dendritic and B cells), and costimulation by CD40/CD40 ligand and CD28/B7. It is maximal at 2‑5 days post activation, or 4 hours post reactivation of memory T cells (3‑6). Human OX40 is a 48 kDa type I transmembrane glycoprotein with a 28 amino acid (aa) signal sequence, a 185 aa extracellular domain (ECD) that has four TNFR-Cys repeats and an O-glycosylated hinge region, a 20 aa transmembrane segment, and a 41 aa cytoplasmic domain (3). The ECD of human OX40 shows 71%, 68%, 67%, 64% and 64% aa identity with feline, canine, rabbit, mouse and rat OX40 ECD, respectively. Engagement of OX40 on activated CD4+ T cells by OX40 ligand on activated dendritic cells promotes T cell survival and proliferation, prolongs the immune response, and enhances the number of cells making the transition from effector to memory T cells (1‑6). OX40 signal transduction includes binding TNF receptor-associated factors (TRAFs), and activating NF kappa B and PI3 kinase to enhance expression of cytokines, antiapoptotic Bcl-2 family members, survivin and the chemokine receptor CXCR5 (5‑8). CXCR5 promotes T cell migration to germinal centers to deliver B cell help (5). Studies using knockout or transgenic mice, and agonistic or blocking antibodies, show that OX40/OX40L interaction is critical for establishing or reactivating memory T cells and breaking immune tolerance (9). Blockade of OX40 engagement is efficacious in animal models of allergic airway inflammation, graft-versus-host disease and autoimmune disease (10‑14).
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