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4767
NF-κB p65 Antibody Sampler Kit

NF-κB p65 Antibody Sampler Kit #4767

Western Blotting Image 1

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).

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Western Blotting Image 2

Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with constructs expressing Myc/DDK-tagged human NF-κB p65 (hNF-κB p65; +) and HA-tagged human p300 (hp300-HA; +), using Acetyl-NF-κB p65 (Lys310) (D2S3J) Rabbit mAb (upper) or NF-κB p65 (D14E12) XP® Rabbit mAb #8242 (lower).

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Western Blotting Image 3

Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-) or SignalSilence® NF-κB p65 siRNA I #6261 (+), using NF-κB p65 (L8F6) Mouse mAb (upper) or α-Tubulin (11Η10) Rabbit mAb #2125 (lower). The NF-κB p65 (L8F6) Mouse mAb confirms silencing of NF-κB p65 expression, while the α-Tubulin (11Η10) Rabbit mAb is used as a loading control.

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Chromatin IP-seq Image 4

Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 HeLa cells treated with hTNF-α #8902 (30 ng/ml, 1 hr) and 5 μl of NF-κB p65 (D14E12) XP® Rabbit mAb, using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared from 5 ng enriched ChIP DNA using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina®, and sequenced on the Illumina NextSeq. 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.

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Flow Cytometry Image 5

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).

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Western Blotting Image 6

Western blot analysis of extracts from various cell lines using NF-κB p65 (D14E12) XP® Rabbit mAb.

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Western Blotting Image 7

Western blot analysis of extracts from HeLa cells treated for 5 minutes with TNF-alpha #2169 (20 ng/ml), Calyculin A #9902 (50 nM), or both compounds, using Phospho-NF-kappaB p65 (Ser468) Antibody (top) or NF-kappaB p65 Antibody #3034 (bottom).

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Western Blotting Image 8

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.

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Western Blotting Image 9

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).

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IP Image 10

Immunoprecipitation of acetyl-NF-κB p65 (Lys310) from 293T cells, cotransfected with Myc/DDK-tagged human NF-κB p65 and HA-tagged human p300, using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or Acetyl-NF-κB p65 (Lys310) (D2S3J) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using Acetyl-NF-κB p65 (Lys310) (D2S3J) Rabbit mAb.

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Western Blotting Image 11

Western blot analysis of extracts from various cell lines using NF-κB p65 (L8F6) Mouse mAb.

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Chromatin IP Image 12

Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 HeLa cells treated with hTNF-α #8902 (30 ng/ml, 1 hr) and either 5 μl of NF-κB p65 (D14E12) XP® Rabbit mAb or 2 μl of 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.

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IHC-P (paraffin) Image 13

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)).

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Flow Cytometry Image 14

Flow cytometric analysis of HeLa cells, untreated (blue) or TNF-α-treated (green), using Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb compared to a nonspecific negative control antibody (red).

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IHC-P (paraffin) Image 15

Immunohistochemical analysis of human chronic cholecystitis tissue using NF-κB p65 (L8F6) Mouse mAb.

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IHC-P (paraffin) Image 16

Immunohistochemical analysis of paraffin-embedded human chronic cholecystitis using NF-κB p65 (D14E12) XP® Rabbit mAb.

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IF-IC 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).

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IHC-P (paraffin) Image 18

Immunohistochemical analysis of paraffin-embedded OVCAR8 cell pellets treated with Human Tumor Necrosis Factor-α (hTNF-α) #8902 (left) or treated with SignalSilence® NF-κB p65 siRNA I #6261 (right), using NF-κB p65 (L8F6) Mouse mAb.

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IHC-P (paraffin) Image 19

Immunohistochemical analysis of paraffin-embedded HeLa cell pellets, untreated (left) or treated with Human Tumor Necrosis Factor-α (hTNF-α) #8902 (right), using NF-κB p65 (L8F6) Mouse mAb.

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IF-IC Image 20

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).

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Flow Cytometry Image 21

Flow cytometric analysis of HeLa cells using NF-κB p65 (L8F6) Mouse mAb (blue) compared to a nonspecific negative control antibody (red).

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IF-IC Image 22

Confocal immunofluorescent analysis of HeLa cells, untreated (left) or treated with Human Tumor Necrosis Factor-α (hTNF-α) #8902 (20 ng/mL, 20 min; right), using NF-κB p65 (L8F6) Mouse mAb (green). Actin filaments were labeled with DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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Chromatin IP Image 23

Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 HeLa cells treated with Human Tumor Necrosis Factor-α (hTNF-α) #8902 (30 ng/ml, 1 hr) and either 10 μl of NF-κB p65 (L8F6) Mouse mAb or 2 μl of 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.

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Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb 3033 20 µl
  • WB
  • IP
  • IF
  • F
H M R Hm Mk Pg 65 Rabbit IgG
Acetyl-NF-κB p65 (Lys310) (D2S3J) Rabbit mAb 12629 20 µl
  • WB
  • IP
H M 65 Rabbit IgG
NF-κB p65 (L8F6) Mouse mAb 6956 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
  • ChIP
H M R Hm Mk Mi B Dg Pg 65 Mouse IgG2b
NF-κB p65 (D14E12) XP® Rabbit mAb 8242 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
  • ChIP
H M R Hm Mk Dg 65 Rabbit IgG
Phospho-NF-κB p65 (Ser468) Antibody 3039 20 µl
  • WB
  • IP
H M R 65 Rabbit 
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 
Anti-mouse IgG, HRP-linked Antibody 7076 100 µl
  • WB
Horse 

The NF-κB p65 Antibody Sampler Kit contains reagents to examine NF-κB p65/RelA phosphorylation at Ser468 and Ser536; acetylation at Lys310; and total p65 levels.

The total NF-κB p65 antibodies recognize endogenous levels of p65 regardless of post-translational modification state such as phosphorylation or acetylation. The phospho-NF-κB p65 antibodies recognize endogenous levels of p65 only when phosphorylated at their indicated target residues. The Acetyl-NF-κB p65 (Lys310) (D2S3J) Rabbit mAb recognizes transfected levels of p65 only when acetylated at Lys310.

The NF-κB p65 (L8F6) Mouse mAb was produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human NF-κB p65. The Acetyl-NF-κB p65 (Lys310) (D2S3J) Rabbit mAb was produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Lys310 of human NF-κB p65 protein. The NF-κB p65 Antibody was produced by immunizing rabbits with a synthetic peptide corresponding to amino acids surrounding Glu498 of human NF-κB p65. The phospho-specific antibodies were produced by immunizing rabbits with synthetic phosphopeptides corresponding to amino acids surrounding the indicated target residues of human NF-κB p65. Polyclonal antibodies were 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, c-Rel, RelB, 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. Rel proteins bind p50 and p52 to form dimeric complexes that bind DNA and regulate transcription. In unstimulated cells, NF-κB is sequestered in the cytoplasm by IκB inhibitory proteins (3-5). NF-κB-activating agents can induce the phosphorylation of IκB proteins, targeting them for rapid degradation through the ubiquitin-proteasome pathway and releasing NF-κB to enter the nucleus where it regulates gene expression (6-8). NIK and IKKα (IKK1) regulate the phosphorylation and processing of NF-κB2 (p100) to produce p52, which translocates to the nucleus (9-11).

RelA/p65 is a subunit of the NF-κB transcription complex, which plays a crucial role in inflammatory and immune responses. The complex is composed of various homodimeric and heterodimeric Rel family member combinations, the activity of which is modulated by post-translational modifications including phosphorylation and acetylation. p65 phosphorylation by PKA and/or MSK1 at Ser276 allows for increased interaction with the transcriptional coactivator p300/CBP to enhance transcriptional activity. NF-κB dimer assembly with IκB, as well as its DNA binding and transcriptional activities, are regulated by p300/CBP acetyltransferases that principally target Lys218, Lys221 and Lys310 (12-14). This process is reciprocally regulated by histone deacetylases (HDACs); several HDAC inhibitors have been shown to activate NF-κB (12-14). T-cell co-stimulation and Calyculin A have both been shown to increase Ser468 phosphorylation (15,16). IKKβ (but not IKKα) and GSK-3β both target this site (16,17), which appears to have a negative regulatory role not involving inhibition of nuclear translocation after TNF-α or IL-1β stimulation (17). p65 phosphorylation at Ser536 regulates activation, nuclear localization, protein-protein interactions, transcriptional activity, and apoptosis (18-22).

  1. Baeuerle, P.A. and Henkel, T. (1994) Annu Rev Immunol 12, 141-79.
  2. Baeuerle, P.A. and Baltimore, D. (1996) Cell 87, 13-20.
  3. Haskill, S. et al. (1991) Cell 65, 1281-9.
  4. Thompson, J.E. et al. (1995) Cell 80, 573-82.
  5. Whiteside, S.T. et al. (1997) EMBO J 16, 1413-26.
  6. Traenckner, E.B. et al. (1995) EMBO J 14, 2876-83.
  7. Scherer, D.C. et al. (1995) Proc Natl Acad Sci USA 92, 11259-63.
  8. Chen, Z.J. et al. (1996) Cell 84, 853-62.
  9. Senftleben, U. et al. (2001) Science 293, 1495-9.
  10. Coope, H.J. et al. (2002) EMBO J 21, 5375-85.
  11. Xiao, G. et al. (2001) Mol Cell 7, 401-9.
  12. Mattioli I et al. (2004) Blood 104, 3302–4
  13. Buss H et al. (2004) J Biol Chem 279, 49571–4
  14. Schwabe RF and Sakurai H (2005) FASEB J 19, 1758–60
  15. Doyle SL et al. (2005) J Biol Chem 280, 23496–501
  16. Buss H et al. (2004) J Biol Chem 279, 55633–43
  17. Ashburner, B.P. et al. (2001) Mol Cell Biol 21, 7065-77.
  18. Mayo, M.W. et al. (2003) J Biol Chem 278, 18980-9.
  19. Chen, L.F. et al. (2002) EMBO J 21, 6539-48.
  20. Sasaki, C.Y. et al. (2005) J Biol Chem 280, 34538-47.
  21. Mattioli, I. et al. (2004) J Immunol 172, 6336-44.
  22. Bae, J.S. et al. (2003) Biochem Biophys Res Commun 305, 1094-8.
Entrez-Gene Id
5970
Swiss-Prot Acc.
Q04206
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.

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