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Analysis of MILLIPLEX® MAP NF[kappa]B signaling using Luminex® xMAP® technology
Author(s) -
Hwang Joseph,
Warmke Tim,
Wiese Rick
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.1031.4
Subject(s) - iκb kinase , phosphorylation , relb , nf κb , iκbα , traf2 , signal transduction , microbiology and biotechnology , nfkb1 , chemistry , kinase , transcription factor , tumor necrosis factor alpha , cancer research , biology , immunology , biochemistry , gene , tumor necrosis factor receptor
NFκB (nuclear factor kappa B) is a transcription factor that plays a crucial regulatory role in both physiological and pathological processes, such as immune responses, inflammation, proliferation, necrosis, apoptosis and tumorigenesis. The main NFκB canonical pathway is composed of its receptor, TNF‐R1/2, adaptor proteins (TRADD, RIP1, and TRAF2), kinases (IKKα/β/γ), inhibitors (IκBα) and the NFκB transcription factor. The NFκB pathway is coordinately regulated by phosphorylation and understanding the role of this pathway in normal physiological processes and in diseases requires the ability to simultaneously measure phosphorylation status of multiple protein targets. In recent years, Luminex® xMAP® bead‐based technology, have enabled the measurement of up to 50 proteins simultaneously. Using the NFκB multiplex panel, we observed increased levels of phosphorylated NFκB, IκB, IKKα, IKKβ, IKKγ and FADD in TNFα treated cells. In contrast, no increase in phosphorylation was observed in cells treated with various other stimuli, including anisomycin, staurosporine, hydrogen peroxide, FasL and CoCl 2 . Modifications in phosphorylation levels were confirmed by the Western blotting using the detection antibodies from the NFκB multiplex panel. The NFκB multiplex panel allowed for simultaneous detection of multiple analytes in a specific, sensitive, and reproducible manner.