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Tytuł:
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Assessment of canonical NF-κB activity in canine diffuse large B-cell lymphoma.
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Autorzy:
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Gaurnier-Hausser A; Department of Professional Studies in the Health Sciences, Drexel University, Philadelphia, PA, 19102, USA.
Mason NJ
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Źródło:
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Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2015; Vol. 1280, pp. 469-504.
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Typ publikacji:
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Journal Article
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Język:
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English
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Imprint Name(s):
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Publication: Totowa, NJ : Humana Press
Original Publication: Clifton, N.J. : Humana Press,
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MeSH Terms:
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Lymphoma, Large B-Cell, Diffuse/*veterinary
NF-kappa B/*metabolism
Animals ; Blotting, Western/methods ; Dogs ; Electrophoretic Mobility Shift Assay ; Enzyme Activation ; Leukocytes, Mononuclear/drug effects ; Leukocytes, Mononuclear/metabolism ; Lymph Nodes/pathology ; Tumor Necrosis Factor-alpha/pharmacology
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Substance Nomenclature:
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0 (NF-kappa B)
0 (Tumor Necrosis Factor-alpha)
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Entry Date(s):
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Date Created: 20150305 Date Completed: 20151120 Latest Revision: 20170714
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Update Code:
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20240104
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DOI:
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10.1007/978-1-4939-2422-6_29
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PMID:
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25736768
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Companion dogs with spontaneous malignancies are clinically relevant models in which to study the corresponding human diseases and potential therapies. In both dogs and people, non-Hodgkin's lymphoma (NHL) is the most common hematopoietic malignancy. Diffuse large B-cell lymphoma (DLBCL) is the most common NHL subtype in dogs and people, sharing similar biologic, behavioral, genetic, and molecular characteristics in both species. One such molecular characteristic is the constitutive activation of the canonical NF-κB pathway, which in health regulates the expression of target genes that control cellular proliferation, survival, and immune and inflammatory responses as well as multidrug resistance. We found that canine and human DLBCL patients share similar NF-κB activity profiles. Using the cell-permeable NBD peptide, which blocks NF-κB signaling, we inhibited constitutive NF-κB activity and induced apoptosis of primary canine malignant B cells in vitro. In addition, we found that NBD peptide administration to dogs with relapsed B-cell lymphoma inhibited the expression of NF-κB target genes and reduced tumor burden. In this chapter, we describe our methods for processing canine malignant lymphoid tissue. We also describe our methods for treating the lymphocytes isolated from this tissue with NBD peptide and evaluating constitutive canonical NF-κB activity in these cells via immunoblot and electrophoretic mobility shift assay (EMSA). We highlight the nuances of working with canine primary cells.