Epitope retrieval with Tris-EDTA pH9.0 is recommended
Application notesIs unsuitable for IP.
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.
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Anti-NF-kB p65 (phospho S536) antibody images
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NF-kB p65 (phospho S536) antibody (ab86299)Kiss T et al., PLoS ONE. 2014;9(8):e104890. Fig #3B.; doi:10.1371/journal.pone.0104890.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat lung tissue labeling NF-kB p65 (phospho S536) with ab86299 at 1/20 dilution. Tissue sections were fixed in 6%formalin and embedded into paraffin, 5 µm thin sections were cut with a microtome. Sections were stained with haematoxylin–eosin. Slides were deparaffinized in xilene, rehydrated in graded ethanol series, and washed in distilled water. Heat induced epitope retrieval was performed by boiling the tissue sections in citrate buffer in a microwave oven at 750 W followed by cooling at room temperature for 20 minutes. Slides were washed in tris buffered saline (TBS) solution (pH = 7,6) followed by blocking of endogenous peroxidase for 10 minutes at room temperature. Slides were washed in TBS. Nonspecific sites were blocked for 10 minutes at room temperature. Without washing, the primary antibody ab86299 was applied. Incubation with the primary antibody was performed for 1 hour at room temperature followed by washing in TBS. Anti-rabbit secondary antibody was applied for 30 minutes at room temperature followed by repeated washing in TBS.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human ovarian carcinoma tissue labelling NF-kB p65 (phospho S536) with ab86299 at 1/200 (1µg/ml) and 1/1000 (0.2µg/ml). Detection: DAB.
Western blot - Anti-NF-kB p65 (phospho S536) antibody (ab86299)
All lanes : Anti-NF-kB p65 (phospho S536) antibody (ab86299) at 0.04 µg/ml
Lane 1 : Jurkat whole cell lysate treated with TNF alpha and
Calyculin A Lane 2 : Jurkat whole cell lysate mock treated
IHC image of NF-kB p65 (phospho S536) staining in Human breast adenocarcinoma formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab86299, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
References for Anti-NF-kB p65 (phospho S536) antibody (ab86299)
This product has been referenced in:
Kang K et al. Carnosic acid slows photoreceptor degeneration in the Pde6b(rd10) mouse model of retinitis pigmentosa. Sci Rep6:22632 (2016).
Read more (PubMed: 26961159) »
Koon HW et al. Probiotic Saccharomyces boulardii CNCM I-745 prevents outbreak-associated Clostridium difficile-associated cecal inflammation in hamsters. Am J Physiol Gastrointest Liver Physiol311:G610-G623 (2016).
Read more (PubMed: 27514478) »