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Phospho-NF-kB p65 (S529) Recombinant Rabbit Monoclonal Antibody [SP07-00]

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Specification

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Catalog# ET1604-27

Phospho-NF-kB p65 (S529) Recombinant Rabbit Monoclonal Antibody [SP07-00]

Application

  • WB

  • IHC-P

  • FC

  • IP

Reactivity

  • Human

Conjugation

  • unconjugated

This product has been cited in peer reviewed publications, see list HERE

Overview

Product Name

Phospho-NF-kB p65 (S529) Recombinant Rabbit Monoclonal Antibody [SP07-00]

Antibody Type

Recombinant Rabbit monoclonal Antibody

Immunogen

Synthetic phospho-peptide corresponding to residues surrounding Ser529 of Human RELA.

Species Reactivity

Human

Validated Applications

WB, IHC-P, FC, IP

Molecular Weight

Predicted band size: 60 kDa

Positive Control

HeLa treated with 100nM Calyculin A and 20ng/mL TNF-α for 15 minutes cell lysate, human lung tissue, human lung squamous cell carcinoma tissue, Daudi.

Conjugation

unconjugated

Clone Number

SP07-00

RRID

AB_3069692

Product Features

Form

Liquid

Concentration

Storage Instructions

Shipped at 4℃. Store at +4℃ short term (1-2 weeks). It is recommended to aliquot into single-use upon delivery. Store at -20℃ long term.

Storage Buffer

1*TBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.

Isotype

IgG

Purification Method

Protein A affinity purified.

Application Dilution

  • WB

  • 1:1,000

  • IHC-P

  • 1:1,000

  • FC

  • 1:50

  • IP

  • Use at an assay dependent concentration.

Target

Function

Proteins encoded by the v-Rel viral oncogene and its cellular homolog, c-Rel, are members of a family of transcription factors that include the two subunits of the transcription factor NκB (p50 and p65) and the Drosophila maternal morphogen, dorsal. Both proteins specifically bind to DNA sequences that are the same or slight variations of the 10 bp κB sequence in the immunoglobulin κ light chain enhancer. This same sequence is also present in a number of other cellular and viral enhancers. The DNA binding activity of NFκB is activated and NFκB is subsequently transported from the cytoplasm to the nucleus in cells exposed to mitogens or growth factors. cDNAs encoding precursors for two distinct proteins of the same size have been described, designated p105 and p100. The p105 precursor contains p50 at its N-terminus and a C-terminal region that when expressed as a separate molecule, designated pdI, binds to p50 and regulates its activity.

Background References

1. Yang K et al. Effect of PLCe gene silencing on inhibiting the cancerous transformation of ulcerative colitis. Exp Ther Med 12:422-426 (2016).

2. Jeon M et al. Elevated IL-1 expression induces invasiveness of triple negative breast cancer cells and is suppressed by zerumbone. Chem Biol Interact 258:126-33 (2016).

Post-translational Modification

Ubiquitinated by RNF182, 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 (By similarity).; 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. Phosphorylation at Ser-276 by RPS6KA4 and RPS6KA5 promotes its transactivation and transcriptional activities.; Reversibly acetylated; the acetylation seems to be mediated by CBP, the deacetylation by HDAC3 and SIRT2. 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 at Lys-310 promotes interaction with BRD4. Acetylation can also lower DNA-binding and results in nuclear export. Interaction with BRMS1 promotes deacetylation of Lys-310. Lys-310 is deacetylated by SIRT2.; S-nitrosylation of Cys-38 inactivates the enzyme activity.; Sulfhydration at Cys-38 mediates the anti-apoptotic activity by promoting the interaction with RPS3 and activating the transcription factor activity.; Sumoylation by PIAS3 negatively regulates DNA-bound activated NF-kappa-B.; Proteolytically cleaved within a conserved N-terminus region required for base-specific contact with DNA in a CPEN1-mediated manner, and hence inhibits NF-kappa-B transcriptional activity.

Subcellular Location

Nucleus, Cytoplasm.

UNIPROT

SwissProt: Q04206 Human

Synonyms

Avian reticuloendotheliosis viral (v rel) oncogene homolog A antibody

MGC131774 antibody

NF kappa B p65delta3 antibody

NFKB3 antibody

Nuclear Factor NF Kappa B p65 Subunit antibody

Nuclear factor NF-kappa-B p65 subunit antibody

Nuclear factor of kappa light polypeptide gene enhancer in B cells 3 antibody

Nuclear factor of kappa light polypeptide gene enhancer in B-cells 3 antibody

OTTHUMP00000233473 antibody

OTTHUMP00000233474 antibody

Expand

Images

  • <span style="font-weight: bold;">☑ Cell treatment (CT)</span><br /><br />Western blot analysis of Phospho-NF-kB p65 (S529) on different lysates with Rabbit anti-Phospho-NF-kB p65 (S529) antibody (<a href="/products/ET1604-27" style="font-weight: bold;text-decoration: underline;">ET1604-27</a>) at 1/1,000 dilution.<br /><br />Lane 1: HeLa cell lysate<br />Lane 2: HeLa treated with 100nM Calyculin A and 20ng/mL TNF-α for 15 minutes cell lysate<br />Lane 3: HeLa treated with 100nM Calyculin A and 20ng/mL TNF-α for 15 minutes cell lysate, then the membrane treated with λpp for 1 hour<br /><br />Lysates/proteins at 20 µg/Lane.<br /><br />Predicted band size: 60 kDa<br />Observed band size: 65 kDa<br /><br />Exposure time: 43 seconds;<br /><br />4-20% SDS-PAGE gel.<br /><br />Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (<a href="/products/ET1604-27" style="font-weight: bold;text-decoration: underline;">ET1604-27</a>) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (<a href="/products/HA1001" style="font-weight: bold;text-decoration: underline;">HA1001</a>) at 1/50,000 dilution was used for 1 hour at room temperature.

    ☑ Cell treatment (CT)

    Western blot analysis of Phospho-NF-kB p65 (S529) on different lysates with Rabbit anti-Phospho-NF-kB p65 (S529) antibody (ET1604-27) at 1/1,000 dilution.

    Lane 1: HeLa cell lysate
    Lane 2: HeLa treated with 100nM Calyculin A and 20ng/mL TNF-α for 15 minutes cell lysate
    Lane 3: HeLa treated with 100nM Calyculin A and 20ng/mL TNF-α for 15 minutes cell lysate, then the membrane treated with λpp for 1 hour

    Lysates/proteins at 20 µg/Lane.

    Predicted band size: 60 kDa
    Observed band size: 65 kDa

    Exposure time: 43 seconds;

    4-20% SDS-PAGE gel.

    Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (ET1604-27) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

  • Immunohistochemical analysis of paraffin-embedded human lung tissue with Rabbit anti-Phospho-NF-kB p65 (S529) antibody (<a href="/products/ET1604-27" style="font-weight: bold;text-decoration: underline;">ET1604-27</a>) at 1/1,000 dilution.<br /><br />The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH<sub>2</sub>O and PBS, and then probed with the primary antibody (<a href="/products/ET1604-27" style="font-weight: bold;text-decoration: underline;">ET1604-27</a>) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

    Immunohistochemical analysis of paraffin-embedded human lung tissue with Rabbit anti-Phospho-NF-kB p65 (S529) antibody (ET1604-27) at 1/1,000 dilution.

    The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1604-27) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

  • Immunohistochemical analysis of paraffin-embedded human lung squamous cell carcinoma tissue with Rabbit anti-Phospho-NF-kB p65 (S529) antibody (<a href="/products/ET1604-27" style="font-weight: bold;text-decoration: underline;">ET1604-27</a>) at 1/1,000 dilution.<br /><br />The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH<sub>2</sub>O and PBS, and then probed with the primary antibody (<a href="/products/ET1604-27" style="font-weight: bold;text-decoration: underline;">ET1604-27</a>) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

    Immunohistochemical analysis of paraffin-embedded human lung squamous cell carcinoma tissue with Rabbit anti-Phospho-NF-kB p65 (S529) antibody (ET1604-27) at 1/1,000 dilution.

    The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (ET1604-27) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

  • Flow cytometric analysis of Phospho-NF-kB p65 (S529) was done on Daudi cells. The cells were fixed, permeabilized and stained with the primary antibody (<a href="/products/ET1604-27" style="font-weight: bold;text-decoration: underline;">ET1604-27</a>, 1/50) (red). After incubation of the primary antibody at room temperature for an hour, the cells were stained with a Alexa Fluor 488-conjugated Goat anti-Rabbit IgG Secondary antibody at 1/1000 dilution for 30 minutes.Unlabelled sample was used as a control (cells without incubation with primary antibody; black).

    Flow cytometric analysis of Phospho-NF-kB p65 (S529) was done on Daudi cells. The cells were fixed, permeabilized and stained with the primary antibody (ET1604-27, 1/50) (red). After incubation of the primary antibody at room temperature for an hour, the cells were stained with a Alexa Fluor 488-conjugated Goat anti-Rabbit IgG Secondary antibody at 1/1000 dilution for 30 minutes.Unlabelled sample was used as a control (cells without incubation with primary antibody; black).

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

Citation

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