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9936
NF-κB Pathway Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit

NF-κB Pathway Antibody Sampler Kit #9936

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Citations (76)
NF-κB Pathway Antibody Sampler Kit: Image 1
Western blot analysis of extracts from various cell lines using IKKα (3G12) Mouse mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
NF-κB Pathway Antibody Sampler Kit: Image 2
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.
NF-κB Pathway Antibody Sampler Kit: Image 3
Western blot analysis of extracts from HeLa and NIH/3T3 cells, untreated or treated with TNF-α (#2169, 20 ng/ml) for 5 minutes, using Phospho-IκBα (Ser32) (14D4) Rabbit mAb (upper), or IκBα (44D4) Rabbit mAb #4812 (lower).
NF-κB Pathway Antibody Sampler Kit: Image 4
Western blot analysis of extracts from HeLa and NIH/3T3 cells, untreated or TNF-α treated (#2169, 20 ng/ml for 5 minutes), using Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb (upper) or NF-κB p65 Antibody #3034 (lower).
NF-κB Pathway Antibody Sampler Kit: Image 5
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-IκBα (Ser32/36) (5A5) Mouse mAb #9246 (upper) and IκBα (L35A5) Mouse mAb (Amino-terminal Antigen) (lower).
NF-κB Pathway Antibody Sampler Kit: Image 6
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.
NF-κB Pathway Antibody Sampler Kit: Image 7
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.
NF-κB Pathway Antibody Sampler Kit: Image 8
Western blot analysis of extracts from various cell lines using NF-κB p65 (D14E12) XP® Rabbit mAb.
NF-κB Pathway Antibody Sampler Kit: Image 9
Western blot analysis of extracts from various cell lines using IKKβ (D30C6) Rabbit mAb.
NF-κB Pathway Antibody Sampler Kit: Image 10
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.
NF-κB Pathway Antibody Sampler Kit: Image 11
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.
NF-κB Pathway Antibody Sampler Kit: Image 12
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-NF-κB p65 (Ser536) (93H1) Rabbit mAb (upper) and NF-κB p65 (C22B4) Rabbit mAb #4764 (lower).
NF-κB Pathway Antibody Sampler Kit: Image 13
Western blot analysis of extracts from HeLa, NIH/3T3 and PC12 cells, using IkBα (L35A5) Mouse mAb (Amino-terminal Antigen).
NF-κB Pathway Antibody Sampler Kit: Image 14
Western blot analysis of extracts from wild-type, IKKα (-/-), and IKKβ (-/-) mouse embryonic fibroblasts (MEFs) using IKKβ (D30C6) Rabbit mAb (upper) and GAPDH (14C10) Rabbit mAb #2118 (lower).
NF-κB Pathway Antibody Sampler Kit: Image 15
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).
NF-κB Pathway Antibody Sampler Kit: Image 16
Immunohistochemical analysis of paraffin-embedded human gall bladder (chronic cholecystitis), using Phospho-IKKα/β (Ser176/180) (16A6) Rabbit mAb.
NF-κB Pathway Antibody Sampler Kit: Image 17
Confocal immunofluorescent analysis of HeLa cells, serum starved (left) or TNF-α treated (#8902 at 20 ng/ml for 20 min, right), using Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb (green). Actin filaments have been labeled with Alexa Fluor® phalloidin 555 (red).
NF-κB Pathway Antibody Sampler Kit: Image 18
Immunohistochemical analysis using NF-κB p65 (D14E12) XP® Rabbit mAb on SignalSlide® NF-κB p65 IHC Controls #12873 (paraffin-embedded HCT116 cells, untreated (left) or treated with hTNF-α #8902 (right)).
NF-κB Pathway Antibody Sampler Kit: Image 19
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.
NF-κB Pathway Antibody Sampler Kit: Image 20
Immunohistochemical analysis of paraffin-embedded human colon carcinoma, untreated (left) or λ phosphatase-treated (right), using Phospho-IKKα/β (Ser176/180) (16A6) Rabbit mAb.
NF-κB Pathway Antibody Sampler Kit: Image 21
Flow cytometric analysis of HeLa cells, untreated (blue) or treated with Human Tumor Necrosis Factor-α (hTNF-α) #8902 and Calyculin A #9902 (20 ng/ml and 100 nM, 15 min; green), using Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
NF-κB Pathway Antibody Sampler Kit: Image 22
Immunohistochemical analysis of paraffin-embedded human leiomyoma, using IκBα (L35A5) Mouse mAb (Amino-terminal Antigen).
NF-κB Pathway Antibody Sampler Kit: Image 23
Immunohistochemical analysis of paraffin-embedded human chronic cholecystitis using NF-κB p65 (D14E12) XP® Rabbit mAb.
NF-κB Pathway Antibody Sampler Kit: Image 24
Immunohistochemical analysis of paraffin-embedded human colon carcinoma, showing cytoplasmic localization, using Phospho-IKKα/β (Ser176/180) (16A6) Rabbit mAb.
NF-κB Pathway Antibody Sampler Kit: Image 25
Immunohistochemical analysis of paraffin-embedded human lung carcinoma, using IκBα (L35A5) Mouse mAb (Amino-terminal Antigen).
NF-κB Pathway Antibody Sampler Kit: Image 26
Confocal immunofluorescent analysis of HT-1080 cells, untreated (left) or treated with hTNF-α #8902 (20 ng/ml, 20 min) (right), using NF-κB p65 (D14E12) XP® Rabbit mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
NF-κB Pathway Antibody Sampler Kit: Image 27
Immunohistochemical analysis of paraffin-embedded human lung (chronic bronchitis), using Phospho-IKKα/β (Ser176/180) (16A6) Rabbit mAb.
NF-κB Pathway Antibody Sampler Kit: Image 28
Immunohistochemical analysis of paraffin-embedded human renal adenocarcinoma, using IκBα (L35A5) Mouse mAb (Amino-terminal Antigen).
NF-κB Pathway Antibody Sampler Kit: Image 29
Flow cytometric analysis of HeLa cells using NF-κB p65 (D14E12) XP® Rabbit mAb (blue) compared to concentration matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (red).
NF-κB Pathway Antibody Sampler Kit: Image 30
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).
NF-κB Pathway Antibody Sampler Kit: Image 31
Confocal immunofluorescent analysis of HeLa cells, untreated (left), or TNF-α-treated (#8902, 10 ng/ml for 15 min, right) using IκBα (L35A5) Mouse mAb (Amino-terminal Antigen) (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
NF-κB Pathway Antibody Sampler Kit: Image 32
Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells treated with hTNF-α #8902 (30 ng/ml, 1 hr) and NF-κB p65 (D14E12) XP® Rabbit mAb, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figure shows binding across IL-8, a known target gene of NFκB (see additional figure containing ChIP-qPCR data). For additional ChIP-seq tracks, please download the product data sheet.
NF-κB Pathway Antibody Sampler Kit: Image 33
Flow cytometric analysis of HeLa cells, using IκBα (L35A5) Mouse mAb (Amino-terminal Antigen) (blue) compared to a nonspecifc negative control antibody (red).
NF-κB Pathway Antibody Sampler Kit: Image 34
Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells treated with hTNF-α #8902 (30 ng/ml, 1 hr) and NF-κB p65 (D14E12) XP® Rabbit mAb, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figure shows binding across chromosome 4 (upper), including IL-8 (lower), a known target gene of NFκB (see additional figure containing ChIP-qPCR data).
NF-κB Pathway Antibody Sampler Kit: Image 35
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.
NF-κB Pathway Antibody Sampler Kit: Image 36
Chromatin immunoprecipitations were performed with cross-linked chromatin from HeLa cells treated with hTNF-α #8902 (30 ng/ml, 1 hr) and either NF-κB p65 (D14E12) XP® Rabbit mAb or Normal Rabbit IgG #2729 using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified by Real-Time PCR using SimpleChIP® Human IκBα Promoter Primers #5552, human IL-8 promoter primers, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.
NF-κB Pathway Antibody Sampler Kit: Image 37
CUT&RUN was performed with HeLa cells treated with hTNF-α #8902 (30 ng/ml, 1 hr) and NF-κB p65 (D14E12) XP® Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figure shows binding across LAMC2, a known target gene of NF-κB p65 (see additional figure containing CUT&RUN-qPCR data).
NF-κB Pathway Antibody Sampler Kit: Image 38
CUT&RUN was performed with HeLa cells treated with hTNF-α #8902 (30 ng/ml, 1 hr) and NF-κB p65 (D14E12) XP® Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA Libraries were prepared using SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795. The figures show binding across chromosome 1 (upper), including LAMC2 (lower), a known target gene of NF-κB p65 (see additional figure containing CUT&RUN-qPCR data).
NF-κB Pathway Antibody Sampler Kit: Image 39
CUT&RUN was performed with HeLa cells treated with hTNF-α #8902 (30 ng/ml, 1 hr) and either NF-κB p65 (D14E12) XP® Rabbit mAb or Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN) #66362, using CUT&RUN Assay Kit #86652. The enriched DNA was quantified by real-time PCR using human LAMC2 upstream primers, and human ITM2A upstream primers. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.
To Purchase # 9936T
Product # Size Price
9936T
1 Kit  (7 x 20 microliters) $ 514

Product Includes Quantity Applications Reactivity MW(kDa) Isotype
IKKα (3G12) Mouse mAb 11930 20 µl
  • WB
  • IF
  • F
H Mk 85 Mouse IgG1
IKKβ (D30C6) Rabbit mAb 8943 20 µl
  • WB
  • IP
H M R Mk 87 Rabbit IgG
Phospho-IKKα/β (Ser176/180) (16A6) Rabbit mAb 2697 20 µl
  • WB
  • IHC
  • F
H M R Mk 85 IKK-alpha 87 IKK-beta Rabbit IgG
Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb 3033 20 µl
  • WB
  • IP
  • IF
  • F
H M R Hm Mk Pg 65 Rabbit IgG
IκBα (L35A5) Mouse mAb (Amino-terminal Antigen) 4814 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
H M R Mk B Pg 39 Mouse IgG1
Phospho-IκBα (Ser32) (14D4) Rabbit mAb 2859 20 µl
  • WB
  • IP
H M R Mk 40 Rabbit IgG
NF-κB p65 (D14E12) XP® Rabbit mAb 8242 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
  • ChIP
  • C&R
H M R Hm Mk Dg 65 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 
Anti-mouse IgG, HRP-linked Antibody 7076 100 µl
  • WB
Horse 

Product Description

The NF-κB Pathway Antibody Sampler Kit contains reagents to examine the activation state and total protein levels of key proteins in the NF-κB pathway: IKKα, IKKβ, NF-κB p65/RelA, and IκBα. The kit contains enough primary and secondary antibodies to perform two Western blot experiments per primary antibody.
MW (kDa) 40 IkappaB-alpha. 65 p65/RelA. 85 IKKalpha. 87 IKKbeta.

Specificity / Sensitivity

Phospho-IKKα/β, phospho-NF-κB p65, and phospho-IκBα antibodies recognize endogenous levels of IKKα/β, p65, and IκB-α, respectively, only when phosphorylated at the indicated residues. They do not cross-react with other family members at physiological levels. Total IKKα, IKKβ, p65, and IκBα antibodies recognize endogenous levels of their respective targets regardless of phosphorylation state and do not cross-react with other family members at physiological levels.

Source / Purification

Monoclonal antibodies are produced by immunizing animals with recombinant human proteins or synthetic peptides.

Background

The transcriptional nuclear factor κB (NF-κB)/Rel transcription factors are present in the cytosol in an inactive state, complexed with the inhibitory IκB proteins. Activation occurs via phosphorylation of IκBα at Ser32 and Ser36, resulting in the ubiquitin-mediated proteasome-dependent degradation of IκBα and the release and nuclear translocation of active NF-κB dimers. The regulation of IκBβ and IκBε is similar to that of IκBα, however, the phosphorylation and degradation of these proteins occurs with much slower kinetics. Phosphorylation of IκBβ occurs at Ser/Thr19 and Ser23, while IκBε can be phosphorylated at Ser18 and Ser22. The key regulatory step in this pathway involves activation of a high molecular weight IkappaB kinase (IKK) complex, consisting of three tightly associated IKK subunits. IKKα and IKKβ serve as the catalytic subunits of the kinase. Activation of IKK depends on phosphorylation at Ser177 and Ser181 in the activation loop of IKKβ (176 and 180 in IKKα). NF-κB-inducing kinase (NIK), TANK-binding kinase 1 (TBK1), and its homolog IKKε (IKKi), phosphorylate and activate IKKα and IKKβ.
The NF-κB family of transcription factors is comprised of five proteins in mammals, p65/RelA, c-Rel, RelB, NF-κB1 (p105/p50) and NF-κB2 (p100/p52). p105 and p100 are proteolytically processed to produce p50 and p52, respectively. The 50 kDa active form is produced through proteolytic processing following IKK-mediated phosphorylation of p105 at multiple sites (Ser922, 924, 928 and 933), while p100's processing to p52 is induced by phosphorylation of Ser864 and Ser868. The p50 and p52 products form dimeric complexes with Rel proteins, which are then able to bind DNA and regulate transcription. Phosphorylation of p65/RelA at Ser276 by PKA C and MSK1 enhances transcriptional activity. p65 phosphorylation at Ser536 regulates activation, nuclear localization, protein-protein interactions, and transcriptional activity. PMA-induced NF-κB transcriptional activity is dependent on the region of p65 containing the potential phosphorylation sites Ser457, Thr458, Thr464 and Ser468. Phosphorylation of Ser468 by GSK-3β inhibits basal p65 activity.
  1. Yamamoto, Y. and Gaynor, R.B. (2004) Trends Biochem. Sci. 29, 72-79.
  2. Ghosh, S. and Karin, M. (2002) Cell 109, S81-S96.
  3. Viatour, P. et al. (2005) Trends Biochem. Sci. 30, 43-52.
  4. Ho, C. et al. (2016) PLOS One 10,1-22.
  5. Beyaz S. et al. (2016) Nature 531, 53-58.

Limited Uses

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Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST's products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST's Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

For Research Use Only. Not For Use In Diagnostic Procedures.
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