Flow cytometric analysis of THP-1 cells, untreated (blue) and with TPA and LPS (green) using IKK-α (Ser176/Ser180) phosphate Rabbit mAb. Anti-rabbit IgG (H+L), F(ab')2 Fragment (PE Conjugate) #8885 was used as a secondary antibody.
Flow cytometric analysis of HCT 116 cells using IKKα (3G12) Mouse mAb (blue) compared to concentration-matched Mouse (G3A1) mAb IgG1 Isotype Control #5415 (red). Anti-mouse IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4408 was used as a secondary antibody.
Western blot analysis of extracts from HeLa cells transfected with wild-type or mutant NF-κB2 p100 (SS866/870AA) and with or without NIK, using Phospho-NF-κB2 p100 (Ser866/870) Antibody or total NF-κB2 p100 Antibody #4882. The p100 constructs were generously provided by Dr. Warner Greene of the Gladstone Institute of Virology and Immunology, Dr. Shao-Cong Sun of The Pennsylvania State University College of Medicine, and Dr. Gutian Xiao of Rutgers, The State University of New Jersey.
Western blot analysis of extracts from THP-1, L929, C6, and COS cells, using NF-kappaB2 p100 Antibody.
Western blot analysis of extracts from various cell lines, untreated or treated with 10uM MG132, using NIK Antibody #4994.
Western blot analysis of extracts from Raji, THP-1 and BaF3 cells using RelB (C1E4) Rabbit mAb.
Western blot analysis of extracts from THP1 and Ramos cells, using TRAF2 Antibody.
Western blot analysis of extracts from COS-7 cells, untransfected (-) or transfected with human TRAF3 construct (+) using TRAF3 Antibody.
After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.
Confocal immunofluorescent analysis of HCT 116 (high expression; left) and IGROV-1 (low expression; right) cells using IKKα (3G12) Mouse mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Western blot analysis of extacts from HeLa cells, untransfected or transfected with HA-NIK, using NIK Antibody #4994.
Western blot analysis of extracts from various cell lines using TRAF3 Antibody.
Immunohistochemical analysis of paraffin-embedded human gall bladder (chronic cholecystitis), using Phospho-IKKα/β (Ser176/180) (16A6) Rabbit mAb.
Western blot analysis of extracts from various cell lines using IKKα (3G12) Mouse mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Immunohistochemical analysis of paraffin-embedded human colon carcinoma, untreated (left) or λ phosphatase-treated (right), using Phospho-IKKα/β (Ser176/180) (16A6) Rabbit mAb.
Western blot analysis of HeLa cell extracts (lane 1) or IKKα knock-out HeLa cells (lane 2), using IKKα (3G12) Mouse mAb #11930 (upper), or β-actin (13E5) Rabbit mAb #4970 (lower). The absence of signal in the knockout HeLa cells confirms specificity of the antibody for IKKα.
Immunohistochemical analysis of paraffin-embedded human colon carcinoma, showing cytoplasmic localization, using Phospho-IKKα/β (Ser176/180) (16A6) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human lung (chronic bronchitis), using Phospho-IKKα/β (Ser176/180) (16A6) Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human breast carcinoma, using Phospho-IKKα/β (Ser176/180) (16A6) Rabbit mAb in the presence of control peptide (left) or Phospho-IKK-alpha/beta (Ser176/180) Blocking Peptide #1023 (right).
Western blot analysis of extracts from THP-1 cells, differentiated with TPA (#9905, 80 nM for 24h) and treated with 1 μg/ml LPS for the indicated times, using Phospho-IKKα/β (Ser176/180) (16A6) Rabbit mAb.
Western blot analysis of extracts from TNF-alpha and Calyculin A treated HeLa and NIH/3T3 cells, using Phospho-IKKα/β (Ser176/180) (16A6) Rabbit mAb.
|Phospho-IKKα/β (Ser176/180) (16A6) Rabbit mAb 2697||20 µl||
||H M R Mk||85 IKK-alpha 87 IKK-beta||Rabbit IgG|
|IKKα (3G12) Mouse mAb 11930||20 µl||
||H Mk||85||Mouse IgG1|
|Phospho-NF-κB2 p100 (Ser866/870) Antibody 4810||20 µl||
|NF-κB2 p100/p52 Antibody 4882||20 µl||
||H M R Mk||52 (mature). 120 (precursor).||Rabbit|
|NIK Antibody 4994||20 µl||
|RelB (C1E4) Rabbit mAb 4922||20 µl||
||H M R Mk||70||Rabbit IgG|
|TRAF2 Antibody 4712||20 µl||
||H M R Mk||53||Rabbit|
|TRAF3 Antibody 4729||20 µl||
||H M R Mk||62||Rabbit|
|Anti-rabbit IgG, HRP-linked Antibody 7074||100 µl||
|Anti-mouse IgG, HRP-linked Antibody 7076||100 µl||
This kit contains reagents to examine the activation state and total protein levels of key components in the noncanonical NF-κB pathway: TRAF2, TRAF3, NIK, IKKα, p100, and RelB.
The Phospho-IKKα/β (Ser176/180) (16A6) Rabbit mAb detects IKKα only when phosphorylated at Ser176/180 and IKKβ only when phosphorylated at Ser177/181. The Phospho-NF-κB2 p100 (Ser866/870) Antibody detects transfected levels of NF-κB2 p100 only when phosphorylated at Ser866/870. The TRAF2, TRAF3, IKKα, RelB, and p100/p52 antibodies detect endogenous levels of total protein of their respective targets. The NIK antibody detects transfected levels of NIK regardless of modification state.
Antibodies to phospho-IKKα/ß (Ser176/180) and phospho-p100 (Ser866/870) are produced by immunizing rabbits with synthetic phosphopeptides corresponding to amino acids surrounding the indicated residues of human IKKα and NFκB2 p100, respectively. Antibodies to TRAF2, TRAF3, NIK,, RelB, and p100/p52 are produced by immunizing rabbits with synthetic peptides corresponding to amino acids at the carboxy terminus of TRAF2, in a central region of TRAF3, adjacent to Gly659 of human NIK, surrounding Ser424 of human RelB, and at the amino terminus of human p100/p52, respectively. Monoclonal antibody to IKKα is produced by immunizing animals with a recombinant protein specific to a fragment of human IKKα protein. Polyclonal antibodies are purified by Protein A and peptide affinity chromatography.
Transcription factors of the nuclear factor κB (NF-κB)/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-κB1 (p105/p50) and NF-κB2 (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-κB family members, p52 tends to form dimers primarily with RelB. A plethora of stimuli such TNFα and LPS induce the canonical NF-κB pathway, characterized by the activation of the classical IκB Kinase (IKK) complex (containing IKKα, IKKβ, IKKγ, and ELKS), which then phosphorylates inhibitory IκB 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-κB2 p100 to p52, a process regulated by the NF-κB Inducing Kinase (NIK) and IKKα, but not IKKβ or IKKγ (4-6). NIK phosphorylates IKKα at Ser176/180 (6) and p100 at Ser866/870, then recruits IKKα to p100 where IKKα phosphorylates additional residues in the N- and 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-induced degradation is a key regulatory point in the pathway (13). This pathway is required for B cell maturation and activation, proper architecture of peripheral lymphoid tissue, and safeguards against autoimmunity (14).
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