Synthetic peptide (the amino acid sequence is considered to be commercially sensitive) within Human NF-kB p65 (phospho S536). The exact sequence is proprietary. (Peptide available as ab202905)
WB: HeLa and Daudi cell lysate treated with Calyculin A + TNF-alpha.
IP: Daudi cell lysate treated with Calyculin A + TNF-alpha.
We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated ‘PUR’ on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.
A trial size is available to purchase for this antibody.
Mouse, Rat: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information.
Produced using Abcam's RabMAb® technology. RabMAb® technology is covered by the following U.S. Patents, No. 5, 675, 063 and/or 7, 429, 487.
Storage instructionsShipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle.
Application notesIs unsuitable for Flow Cyt,ICC/IF or IHC-P.
FunctionNF-kappa-B is a pleiotropic transcription factor which is present in almost all cell types and is involved in many biological processed such as inflammation, immunity, differentiation, cell growth, tumorigenesis and apoptosis. NF-kappa-B is a homo- or heterodimeric complex formed by the Rel-like domain-containing proteins RELA/p65, RELB, NFKB1/p105, NFKB1/p50, REL and NFKB2/p52 and the heterodimeric p65-p50 complex appears to be most abundant one. The dimers bind at kappa-B sites in the DNA of their target genes and the individual dimers have distinct preferences for different kappa-B sites that they can bind with distinguishable affinity and specificity. Different dimer combinations act as transcriptional activators or repressors, respectively. NF-kappa-B is controlled by various mechanisms of post-translational modification and subcellular compartmentalization as well as by interactions with other cofactors or corepressors. NF-kappa-B complexes are held in the cytoplasm in an inactive state complexed with members of the NF-kappa-B inhibitor (I-kappa-B) family. In a conventional activation pathway, I-kappa-B is phosphorylated by I-kappa-B kinases (IKKs) in response to different activators, subsequently degraded thus liberating the active NF-kappa-B complex which translocates to the nucleus. NF-kappa-B heterodimeric p65-p50 and p65-c-Rel complexes are transcriptional activators. The NF-kappa-B p65-p65 complex appears to be involved in invasin-mediated activation of IL-8 expression. The inhibitory effect of I-kappa-B upon NF-kappa-B the cytoplasm is exerted primarily through the interaction with p65. p65 shows a weak DNA-binding site which could contribute directly to DNA binding in the NF-kappa-B complex. Associates with chromatin at the NF-kappa-B promoter region via association with DDX1.
Domainthe 9aaTAD motif is a transactivation domain present in a large number of yeast and animal transcription factors.
Post-translational modificationsUbiquitinated, leading to its proteasomal degradation. Degradation is required for termination of NF-kappa-B response. Monomethylated at Lys-310 by SETD6. Monomethylation at Lys-310 is recognized by the ANK repeats of EHMT1 and promotes the formation of repressed chromatin at target genes, leading to down-regulation of NF-kappa-B transcription factor activity. Phosphorylation at Ser-311 disrupts the interaction with EHMT1 without preventing monomethylation at Lys-310 and relieves the repression of target genes. Phosphorylation at Ser-311 disrupts the interaction with EHMT1 and promotes transcription factor activity (By similarity). Phosphorylation on Ser-536 stimulates acetylation on Lys-310 and interaction with CBP; the phosphorylated and acetylated forms show enhanced transcriptional activity. Reversibly acetylated; the acetylation seems to be mediated by CBP, the deacetylation by HDAC3. Acetylation at Lys-122 enhances DNA binding and impairs association with NFKBIA. Acetylation at Lys-310 is required for full transcriptional activity in the absence of effects on DNA binding and NFKBIA association. Acetylation can also lower DNA-binding and results in nuclear export. Interaction with BRMS1 promotes deacetylation of 'Lys-310'.
Cellular localizationNucleus. Cytoplasm. Nuclear, but also found in the cytoplasm in an inactive form complexed to an inhibitor (I-kappa-B). Colocalized with RELA in the nucleus upon TNF-alpha induction.
ab76302 (purified) at 1/30 immunoprecipitating NF-kB p65 (phospho S536) in Daudi whole cell lysate treated with calyculin A and tumor necrosis factor-alpha.
Lane 1 (input): Daudi whole cell lysate treated with calyculin A and tumor necrosis factor-alpha (10µg)
Lane 2 (+): ab76302 + Daudi whole cell lysate treated with calyculin A and tumor necrosis factor-alpha.
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab76302 in Daudi whole cell lysate treated with calyculin A and tumor necrosis factor-alpha.
For western blotting, ab131366 VeriBlot for IP (HRP) was used as the secondary antibody (1/1000).
Blocking buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM /TBST.
Dot Blot - Anti-NF-kB p65 (phospho S536) antibody [EP2294Y] (ab76302)
Dot blot analysis of INF- kB p65 (phospho S536) phospho peptide (Lane 1) and NF- kB p65 non-phospho peptide (Lane 2) labeling NF-kB p65 (phospho S536) with ab76302 at a dilution of 1/1000. ab97051 (Peroxidase conjugated goat anti-rabbit IgG) (H+L) at 1/100 000 was used as the secondary antibody.
Blocking and diluting buffer: 5% NFDM/TBST.
Exposure time: 3 minutes.
Western blot - Anti-NF-kB p65 (phospho S536) antibody [EP2294Y] (ab76302)
All lanes : Anti-NF-kB p65 (phospho S536) antibody [EP2294Y] (ab76302) at 1/20000 dilution (unpurified)
Lane 1 : Untreated Daudi cell lysate Lane 2 : Daudi cell lysate treated with Calyculin A + TNF-alpha
Lysates/proteins at 10 µg per lane.
Secondary HRP labelled goat anti-rabbit at 1/1000 dilution
Predicted band size : 65 kDa
References for Anti-NF-kB p65 (phospho S536) antibody [EP2294Y] (ab76302)
This product has been referenced in:
Liu H et al. Atractylenolide I modulates ovarian cancer cell-mediated immunosuppression by blocking MD-2/TLR4 complex-mediated MyD88/NF-?B signaling in vitro. J Transl Med14:104 (2016).
Read more (PubMed: 27118139) »
Kim S et al. The new 4-O-methylhonokiol analog GS12021 inhibits inflammation and macrophage chemotaxis: role of AMP-activated protein kinase a activation. PLoS One10:e0117120 (2015).
Read more (PubMed: 25706552) »