Description
NF-κB is a ubiquitously expressed transcription factor that regulates many cytokine and Ig genes. It is involved in immune, inflammatory, viral, and acute phase responses. In most cells, NF-κB is sequestered in an inactive cytoplasmic form via interactions with the inhibitory proteins IκBα, IκBβ, and IκBε. Stimulation induces the release, activation, and nuclear translocation of NF-κB. Release of NF-κB results from the phosphorylation and proteolytic degradation of the IκB proteins. Two cytokine-inducible IκB kinases (IKKα and IKKβ) phosphorylate and target the IκB proteins for degradation via the ubiquitin pathway. IKKγ/NEMO, a third member of the IKK complex, functions as a regulatory subunit and interacts directly with IKKβ. TBK1 (TANK-binding kinase 1, also known as T2K or NAK), a protein of 729 amino acids, is another member of the IKK family of kinases regulating NF-κB downstream of the tumor necrosis factor and Toll-like receptor pathways. TBK1 forms a complex with the adaptor proteins TANK (TRAF-associated NF-κB activator) and TRAF2 (TNF-receptor-associated factor 2), and this oligomer is required for activation and phosphorylation of TBK1 at serine 172 (S172). The J133-1171 monoclonal antibody specifically recognizes the phosphorylated S172 form of the human TBK1 protein, TBK1 (pS172). The specificity of the J133-1171 antibody was validated by Western blot analyses of cellular lysates prepared from a cell line co-transfected with TBK1 and IRF-7. The analysis revealed positive reactivity by detection of a band corresponding to TBK1 (pS172). The co-transfected cell line was used since Western blot detection of endogenous TBK1 (pS172) in activated untransfected cell lines (eg, HeLa cells activated with PMA or A431 cells activated with platelet-derived growth factor) was not possible. In the transfectants, the over-expression of TBK1 is necessary for phosphorylation of IRF-7, but the presence of IRF-7 does not affect TBK1 expression or phosphorylation. We confirmed that the J133-1171 antibody does not crossreact with IRF-7 by ELISA.
The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. Store undiluted at 4°C.
1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
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