Transcription factors of the nuclear factor kB (NF-kB)/Rel family play a pivotal role in inflammatory and immune responses (1,2). There are five family members in mammals: RelA, RelB, c-Rel, NF-kB1 (p105/p50) and NF-kB2 (p100/p52). Both p105 and p100 are proteolytically processed by the proteasome to produce p50 and p52, respectively. The p50 and p52 products form dimeric complexes with Rel proteins. While p50 associates with many of the NF-kB family members, p52 tends to form dimers primarily with RelB. A plethora of stimuli such TNFa and LPS induce the canonical NF-kB pathway, characterized by the activation of the classical IkB Kinase (IKK) complex (containing IKKa, IKKb, IKKg, and ELKS), which then phosphorylates inhibitory IkB molecules, targeting them for rapid degradation through a ubiquitin-proteasome pathway (3). The noncanonical pathway, triggered by BAFF, CD40L, and certain other stimuli, is based on the inducible phosphorylation and proteasome-mediated partial degradation of NF-kB2 p100 to p52, a process regulated by the NF-kB Inducing Kinase (NIK) and IKKa, but not IKKb or IKKg (46). NIK phosphorylates IKKa at Ser176/180 (6) and p100 at Ser866/870, then recruits IKKa to p100 where IKKa phosphorylates additional residues in the Nand C-terminus (8), leading to the ubiquitination and processing of p100 (9). The TNF Receptor Associated Factor molecules TRAF2 and TRAF3 have been shown to be negative regulators of the noncanonical pathway (10, 11), and their differential binding to receptors may also play a role in determining whether transduced signals activate the canonical pathway, noncanonical pathway, or both (12). TRAF3 promotes the rapid turnover of NIK in resting cells, and its activation-
Suitable for use in Western Blot. Other applications not tested.
Western Blot: 1:1000
Optimal dilutions to be determined by the researcher.
Storage and Stability:
For long-term storage, aliquot and store at -20 degrees C. Aliquots are stable for at least 6 months at -20 degrees C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.