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#4767 NF-κB p65 Antibody Sampler Kit

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2019年3月20日11時40分 現在
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NF-kB p65 製品一覧

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キット内容 容量 用途 種交差性 ホモロジー† 検出分子量 アイソタイプ
Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb #3033 20 µl WB, IP, IF-IC, F H, M, R, Hm, Mk, Pg Dg 65 Rabbit IgG
Acetyl-NF-κB p65 (Lys310) (D2S3J) Rabbit mAb #12629 20 µl WB, IP H, M Mk 65 Rabbit IgG
NF-κB p65 (L8F6) Mouse mAb #6956 20 µl WB, IP, IHC-P, IF-IC, 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-P, IF-IC, F, ChIP, ChIP-seq 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

†Species predicted to react based on 100% sequence homology.

Applications Key: W=Western Blotting, IP=Immunoprecipitation, IF-IC=Immunofluorescence (Immunocytochemistry), F=Flow Cytometry, IHC-P=Immunohistochemistry (Paraffin), ChIP=Chromatin IP, ChIP-seq=Chromatin IP-seq
Reactivity Key: H=Human, M=Mouse, R=Rat, Hm=Hamster, Mk=Monkey, Pg=Pig, Mi=Mink, B=Bovine, Dg=Dog

貯法
-20℃
※括弧付きの動物種は配列が100%相同であるため反応すると推定されます。
社内データ

Western Blotting

Western Blotting

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.

Western Blotting

Western Blotting

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).

Western Blotting

Western Blotting

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).


Western Blotting

Western Blotting

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

Flow Cytometry

Flow Cytometry

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).

Western Blotting

Western Blotting

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).


Chromatin IP-seq

Chromatin IP-seq

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.

Western Blotting

Western Blotting

Western blot analysis of HeLa cell extracts, untreated (-) or NF-κB p65 knock-out (+), using NF-κB p65 (L8F6) Mouse mAb #6956 (upper) or β-actin (13E5) Rabbit mAb #4970 (lower).

Western Blotting

Western Blotting

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).


Western Blotting

Western Blotting

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.

Chromatin IP

Chromatin IP

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.

IP

IP

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.


IHC-P (paraffin)

IHC-P (paraffin)

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)).

Flow Cytometry

Flow Cytometry

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).

Western Blotting

Western Blotting

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


IHC-P (paraffin)

IHC-P (paraffin)

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

IF-IC

IF-IC

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).

IHC-P (paraffin)

IHC-P (paraffin)

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


IHC-P (paraffin)

IHC-P (paraffin)

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.

IF-IC

IF-IC

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).

IHC-P (paraffin)

IHC-P (paraffin)

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.


Flow Cytometry

Flow Cytometry

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

IF-IC

IF-IC

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).

Chromatin IP

Chromatin IP

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


バックグラウンド

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).

使用例
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関連製品
3017   NF-κB2 p100/p52 (18D10) Rabbit mAb
3035   NF-κB1 p105/p50 Antibody
4717   NF-κB1 p105 Antibody
4766   NF-κB Family Member Antibody Sampler Kit
4806   Phospho-NF-κB p105 (Ser933) (18E6) Rabbit mAb
4808   Phospho-NF-κB p105 (Ser933) (178F3) Rabbit mAb (IHC Specific)
4810   Phospho-NF-κB2 p100 (Ser866/870) Antibody
4882   NF-κB2 p100/p52 Antibody
4922   RelB (C1E4) Rabbit mAb
9243   NF-κB Control Cell Extracts
9936   NF-κB Pathway Sampler Kit

Illumina is a registered trademark of Illumina, Inc.
DRAQ5 is a registered trademark of Biostatus Limited.
XP is a registered trademark of Cell Signaling Technology, Inc.
Alexa Fluor is a registered trademark of Life Technologies Corporation.
LumiGLO is a registered trademark of Kirkegaard & Perry Laboratories.
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.

本製品は試験研究用です。

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