产品: 磷酸化 NF-kB p65 (Ser536) 抗体
货号: AF2006
描述: Rabbit polyclonal antibody to Phospho-NF-kB p65 (Ser536)
应用: WB IHC IF/ICC IP
反应: Human, Rat, Monkey
预测: Mouse, Pig, Bovine, Horse, Sheep, Dog
分子量: 65 kDa; 60kD(Calculated).
蛋白号: Q04206
RRID: AB_2834435

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产品描述

来源:
Rabbit
应用:
WB 1:500-1:2000, IHC 1:50-1:500, IP 1:100-1:500, IF/ICC 1:200
*The optimal dilutions should be determined by the end user.
*Tips:

WB: 适用于变性蛋白样本的免疫印迹检测. IHC: 适用于组织样本的石蜡(IHC-p)或冰冻(IHC-f)切片样本的免疫组化/荧光检测. IF/ICC: 适用于细胞样本的荧光检测. ELISA(peptide): 适用于抗原肽的ELISA检测.

反应:
Human,Rat,Monkey
预测:
Mouse(%), Pig(100%), Bovine(91%), Horse(91%), Sheep(91%), Dog(100%)
克隆:
Polyclonal
特异性:
Phospho-NF-kB p65 (Ser536) Antibody detects endogenous levels of NF-kB p65 only when phosphorylated at Serine 536.
RRID:
AB_2834435
引用格式: Affinity Biosciences Cat# AF2006, RRID:AB_2834435.
偶联:
Unconjugated.
纯化:
The antibody is from purified rabbit serum by affinity purification via sequential chromatography on phospho-peptide and non-phospho-peptide affinity columns.
保存:
Rabbit IgG in phosphate buffered saline , pH 7.4, 150mM NaCl, sodium azide and glycerol. Store at -20 °C. Stable for 12 months from date of receipt.
别名:

展开/折叠

Avian reticuloendotheliosis viral (v rel) oncogene homolog A; MGC131774; NF kappa B p65delta3; NFKB3; Nuclear Factor NF Kappa B p65 Subunit; Nuclear factor NF-kappa-B p65 subunit; Nuclear factor of kappa light polypeptide gene enhancer in B cells 3; Nuclear factor of kappa light polypeptide gene enhancer in B-cells 3; OTTHUMP00000233473; OTTHUMP00000233474; OTTHUMP00000233475; OTTHUMP00000233476; OTTHUMP00000233900; p65; p65 NF kappaB; p65 NFkB; relA; TF65_HUMAN; Transcription factor p65; v rel avian reticuloendotheliosis viral oncogene homolog A (nuclear factor of kappa light polypeptide gene enhancer in B cells 3 (p65)); V rel avian reticuloendotheliosis viral oncogene homolog A; v rel reticuloendotheliosis viral oncogene homolog A (avian); V rel reticuloendotheliosis viral oncogene homolog A, nuclear factor of kappa light polypeptide gene enhancer in B cells 3, p65;

抗原和靶标

免疫原:
Uniprot:
基因/基因ID:
描述:
NFKB1 (MIM 164011) or NFKB2 (MIM 164012) is bound to REL (MIM 164910), RELA, or RELB (MIM 604758) to form the NFKB complex. The p50 (NFKB1)/p65 (RELA) heterodimer is the most abundant form of NFKB. The NFKB complex is inhibited by I-kappa-B proteins (NFKBIA, MIM 164008 or NFKBIB, MIM 604495), which inactivate NFKB by trapping it in the cytoplasm.
序列:
MDELFPLIFPAEPAQASGPYVEIIEQPKQRGMRFRYKCEGRSAGSIPGERSTDTTKTHPTIKINGYTGPGTVRISLVTKDPPHRPHPHELVGKDCRDGFYEAELCPDRCIHSFQNLGIQCVKKRDLEQAISQRIQTNNNPFQVPIEEQRGDYDLNAVRLCFQVTVRDPSGRPLRLPPVLSHPIFDNRAPNTAELKICRVNRNSGSCLGGDEIFLLCDKVQKEDIEVYFTGPGWEARGSFSQADVHRQVAIVFRTPPYADPSLQAPVRVSMQLRRPSDRELSEPMEFQYLPDTDDRHRIEEKRKRTYETFKSIMKKSPFSGPTDPRPPPRRIAVPSRSSASVPKPAPQPYPFTSSLSTINYDEFPTMVFPSGQISQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAPKPTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIADMDFSALLSQISS

种属预测

种属预测:

score>80的预测可信度较高,可尝试用于WB检测。*预测模型主要基于免疫原序列比对,结果仅作参考,不作为质保凭据。

Species
Results
Score
Pig
100
Dog
100
Horse
91
Bovine
91
Sheep
91
Xenopus
0
Zebrafish
0
Chicken
0
Rabbit
0
Model Confidence:
High(score>80) Medium(80>score>50) Low(score<50) No confidence

翻译修饰 - Q04206 作为底物

Site PTM Type Enzyme
M1 Acetylation
K37 Methylation
K37 Sumoylation
C38 S-Nitrosylation
S42 Phosphorylation
S45 Phosphorylation
K56 Ubiquitination
K62 Ubiquitination
T71 Phosphorylation
S75 Phosphorylation
K79 Ubiquitination
K93 Ubiquitination
S112 Phosphorylation
K122 Acetylation
K122 Ubiquitination
K123 Acetylation
K123 Ubiquitination
S131 Phosphorylation
T136 Phosphorylation
R174 Methylation
S180 Phosphorylation
R187 Methylation
K195 Ubiquitination
S205 Phosphorylation
K218 Acetylation
K218 Methylation
K218 Ubiquitination
K221 Acetylation
K221 Methylation
S238 Phosphorylation
S240 Phosphorylation
T254 Phosphorylation
S261 Phosphorylation
S269 Phosphorylation
S276 Phosphorylation P11309 (PIM1) , O94806 (PRKD3) , O75676 (RPS6KA4) , P17612 (PRKACA) , O75582 (RPS6KA5)
S281 Phosphorylation
T305 O-Glycosylation
T305 Phosphorylation
Y306 Phosphorylation
T308 Phosphorylation
K310 Acetylation
K310 Methylation
K310 Ubiquitination
S311 Phosphorylation Q05513 (PRKCZ)
K314 Acetylation
K314 Methylation
K314 Ubiquitination
K315 Acetylation
K315 Methylation
K315 Ubiquitination
S316 Phosphorylation P48729 (CSNK1A1)
S319 O-Glycosylation
T322 O-Glycosylation
S337 O-Glycosylation
S337 Phosphorylation
T352 O-Glycosylation
S374 O-Glycosylation
S374 Phosphorylation
S377 O-Glycosylation
T429 Phosphorylation
T435 Phosphorylation P28482 (MAPK1)
S468 Phosphorylation P49841 (GSK3B) , O14920 (IKBKB) , Q14164 (IKBKE)
S472 Phosphorylation
T505 Phosphorylation
S529 Phosphorylation P68400 (CSNK2A1) , P47710 (CSN1S1)
S536 Phosphorylation O15111 (CHUK) , O94806 (PRKD3) , Q9Y6K9 (IKBKG) , Q9UHD2 (TBK1) , Q9HCP0 (CSNK1G1) , Q16566 (CAMK4) , P51812 (RPS6KA3) , Q15418 (RPS6KA1) , Q00534 (CDK6) , O14920 (IKBKB) , P24723 (PRKCH) , Q14164 (IKBKE)
S543 Phosphorylation P68400 (CSNK2A1)
S547 Phosphorylation Q13315 (ATM)

研究背景

功能:

NF-kappa-B is a pleiotropic transcription factor present in almost all cell types and is the endpoint of a series of signal transduction events that are initiated by a vast array of stimuli related to many biological processes 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. The heterodimeric RELA-NFKB1 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. The NF-kappa-B heterodimeric RELA-NFKB1 and RELA-REL complexes, for instance, function as transcriptional activators. 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. The inhibitory effect of I-kappa-B on NF-kappa-B through retention in the cytoplasm is exerted primarily through the interaction with RELA. RELA shows a weak DNA-binding site which could contribute directly to DNA binding in the NF-kappa-B complex. Beside its activity as a direct transcriptional activator, it is also able to modulate promoters accessibility to transcription factors and thereby indirectly regulate gene expression. Associates with chromatin at the NF-kappa-B promoter region via association with DDX1. Essential for cytokine gene expression in T-cells. The NF-kappa-B homodimeric RELA-RELA complex appears to be involved in invasin-mediated activation of IL-8 expression.

翻译修饰:

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.

细胞定位:

Nucleus. Cytoplasm.
Note: Nuclear, but also found in the cytoplasm in an inactive form complexed to an inhibitor (I-kappa-B) (PubMed:1493333). Colocalized with DDX1 in the nucleus upon TNF-alpha induction (PubMed:19058135). Colocalizes with GFI1 in the nucleus after LPS stimulation (PubMed:20547752). Translocation to the nucleus is impaired in L.monocytogenes infection (PubMed:20855622).

Extracellular region or secreted Cytosol Plasma membrane Cytoskeleton Lysosome Endosome Peroxisome ER Golgi apparatus Nucleus Mitochondrion Manual annotation Automatic computational assertionSubcellular location
亚基结构:

Component of the NF-kappa-B p65-p50 complex. Component of the NF-kappa-B p65-c-Rel complex. Homodimer; component of the NF-kappa-B p65-p65 complex. Component of the NF-kappa-B p65-p52 complex. May interact with ETHE1. Binds TLE5 and TLE1. Interacts with TP53BP2. Binds to and is phosphorylated by the activated form of either RPS6KA4 or RPS6KA5. Interacts with ING4 and this interaction may be indirect. Interacts with CARM1, USP48 and UNC5CL. Interacts with IRAK1BP1 (By similarity). Interacts with NFKBID (By similarity). Interacts with NFKBIA. Interacts with GSK3B. Interacts with NFKBIB (By similarity). Interacts with NFKBIE. Interacts with NFKBIZ. Interacts with EHMT1 (via ANK repeats) (By similarity). Part of a 70-90 kDa complex at least consisting of CHUK, IKBKB, NFKBIA, RELA, ELP1 and MAP3K14. Interacts with HDAC3; HDAC3 mediates the deacetylation of RELA. Interacts with HDAC1; the interaction requires non-phosphorylated RELA. Interacts with CBP; the interaction requires phosphorylated RELA. Interacts (phosphorylated at 'Thr-254') with PIN1; the interaction inhibits p65 binding to NFKBIA. Interacts with SOCS1. Interacts with UXT. Interacts with MTDH and PHF11. Interacts with ARRB2. Interacts with NFKBIA (when phosphorylated), the interaction is direct; phosphorylated NFKBIA is part of a SCF(BTRC)-like complex lacking CUL1. Interacts with RNF25. Interacts (via C-terminus) with DDX1. Interacts with UFL1 and COMMD1. Interacts with BRMS1; this promotes deacetylation of 'Lys-310'. Interacts with NOTCH2 (By similarity). Directly interacts with MEN1; this interaction represses NFKB-mediated transactivation. Interacts with AKIP1, which promotes the phosphorylation and nuclear retention of RELA. Interacts (via the RHD) with GFI1; the interaction, after bacterial lipopolysaccharide (LPS) stimulation, inhibits the transcriptional activity by interfering with the DNA-binding activity to target gene promoter DNA. Interacts (when acetylated at Lys-310) with BRD4; leading to activation of the NF-kappa-B pathway. Interacts with MEFV. Interacts with CLOCK (By similarity). Interacts (via N-terminus) with CPEN1; this interaction induces proteolytic cleavage of p65/RELA subunit and inhibition of NF-kappa-B transcriptional activity. Interacts with FOXP3. Interacts with CDK5RAP3; stimulates the interaction of RELA with HDAC1, HDAC2 and HDAC3 thereby inhibiting NF-kappa-B transcriptional activity. Interacts with DHX9; this interaction is direct and activates NF-kappa-B-mediated transcription. Interacts with LRRC25. Interacts with TBX21 (By similarity). Interacts with KAT2A (By similarity). Interacts with ZBTB7A; involved in the control by RELA of the accessibility of target gene promoters. Directly interacts with DDX3X; this interaction may trap RELA in the cytoplasm, impairing nuclear relocalization upon TNF activating signals.

(Microbial infection) Interacts with human respiratory syncytial virus (HRSV) protein M2-1.

(Microbial infection) Interacts with molluscum contagiosum virus MC132.

(Microbial infection) Interacts with herpes virus 8 virus protein LANA1.

蛋白家族:

The transcriptional activation domain 3/TA3 does not participate to the direct transcriptional activity of RELA but is involved in the control by RELA of the accessibility of target gene promoters. Mediates interaction with ZBTB7A.

The transcriptional activation domain 1/TA1 and the transcriptional activation domain 2/TA2 have direct transcriptional activation properties (By similarity). The 9aaTAD motif found within the transcriptional activation domain 2 is a conserved motif present in a large number of transcription factors that is required for their transcriptional transactivation activity (PubMed:17467953).

研究领域

· Cellular Processes > Cell growth and death > Apoptosis.   (View pathway)

· Cellular Processes > Cell growth and death > Cellular senescence.   (View pathway)

· Environmental Information Processing > Signal transduction > MAPK signaling pathway.   (View pathway)

· Environmental Information Processing > Signal transduction > Ras signaling pathway.   (View pathway)

· Environmental Information Processing > Signal transduction > cAMP signaling pathway.   (View pathway)

· Environmental Information Processing > Signal transduction > NF-kappa B signaling pathway.   (View pathway)

· Environmental Information Processing > Signal transduction > HIF-1 signaling pathway.   (View pathway)

· Environmental Information Processing > Signal transduction > Sphingolipid signaling pathway.   (View pathway)

· Environmental Information Processing > Signal transduction > PI3K-Akt signaling pathway.   (View pathway)

· Environmental Information Processing > Signal transduction > TNF signaling pathway.   (View pathway)

· Human Diseases > Drug resistance: Antineoplastic > Antifolate resistance.

· Human Diseases > Endocrine and metabolic diseases > Insulin resistance.

· Human Diseases > Endocrine and metabolic diseases > Non-alcoholic fatty liver disease (NAFLD).

· Human Diseases > Substance dependence > Cocaine addiction.

· Human Diseases > Infectious diseases: Bacterial > Epithelial cell signaling in Helicobacter pylori infection.

· Human Diseases > Infectious diseases: Bacterial > Shigellosis.

· Human Diseases > Infectious diseases: Bacterial > Salmonella infection.

· Human Diseases > Infectious diseases: Bacterial > Pertussis.

· Human Diseases > Infectious diseases: Bacterial > Legionellosis.

· Human Diseases > Infectious diseases: Parasitic > Leishmaniasis.

· Human Diseases > Infectious diseases: Parasitic > Chagas disease (American trypanosomiasis).

· Human Diseases > Infectious diseases: Parasitic > Toxoplasmosis.

· Human Diseases > Infectious diseases: Parasitic > Amoebiasis.

· Human Diseases > Infectious diseases: Bacterial > Tuberculosis.

· Human Diseases > Infectious diseases: Viral > Hepatitis C.

· Human Diseases > Infectious diseases: Viral > Hepatitis B.

· Human Diseases > Infectious diseases: Viral > Measles.

· Human Diseases > Infectious diseases: Viral > Influenza A.

· Human Diseases > Infectious diseases: Viral > Human papillomavirus infection.

· Human Diseases > Infectious diseases: Viral > HTLV-I infection.

· Human Diseases > Infectious diseases: Viral > Herpes simplex infection.

· Human Diseases > Infectious diseases: Viral > Epstein-Barr virus infection.

· Human Diseases > Cancers: Overview > Pathways in cancer.   (View pathway)

· Human Diseases > Cancers: Overview > Transcriptional misregulation in cancer.

· Human Diseases > Cancers: Overview > Viral carcinogenesis.

· Human Diseases > Cancers: Specific types > Pancreatic cancer.   (View pathway)

· Human Diseases > Cancers: Specific types > Prostate cancer.   (View pathway)

· Human Diseases > Cancers: Specific types > Chronic myeloid leukemia.   (View pathway)

· Human Diseases > Cancers: Specific types > Acute myeloid leukemia.   (View pathway)

· Human Diseases > Cancers: Specific types > Small cell lung cancer.   (View pathway)

· Human Diseases > Immune diseases > Inflammatory bowel disease (IBD).

· Organismal Systems > Immune system > Chemokine signaling pathway.   (View pathway)

· Organismal Systems > Aging > Longevity regulating pathway.   (View pathway)

· Organismal Systems > Development > Osteoclast differentiation.   (View pathway)

· Organismal Systems > Immune system > Toll-like receptor signaling pathway.   (View pathway)

· Organismal Systems > Immune system > NOD-like receptor signaling pathway.   (View pathway)

· Organismal Systems > Immune system > RIG-I-like receptor signaling pathway.   (View pathway)

· Organismal Systems > Immune system > Cytosolic DNA-sensing pathway.   (View pathway)

· Organismal Systems > Immune system > IL-17 signaling pathway.   (View pathway)

· Organismal Systems > Immune system > Th1 and Th2 cell differentiation.   (View pathway)

· Organismal Systems > Immune system > Th17 cell differentiation.   (View pathway)

· Organismal Systems > Immune system > T cell receptor signaling pathway.   (View pathway)

· Organismal Systems > Immune system > B cell receptor signaling pathway.   (View pathway)

· Organismal Systems > Nervous system > Neurotrophin signaling pathway.   (View pathway)

· Organismal Systems > Endocrine system > Prolactin signaling pathway.   (View pathway)

· Organismal Systems > Endocrine system > Adipocytokine signaling pathway.

· Organismal Systems > Endocrine system > Relaxin signaling pathway.

文献引用

1). Gut microbial metabolite trimethylamine N-oxide aggravates GVHD by inducing M1 macrophage polarization in mice. BLOOD, 2020 (PubMed: 32291445) [IF=20.3]

Application: IF/ICC    Species: mouse    Sample: BMDMs

Supplemental Figure 7.| TMAO induced NLRP3 expression by enhanced NF-κB nuclear localization. (A) Representative immunofluorescence staining of NF-κB (red: Alexa Fluor 594) in 300µM TMAO-treated BMDMs for 24h.Scale bar=25μm (B) Representative immunofluorescence image of p-NF-κB (red: Alexa Fluor 594) in BMDMs cultured with Triptolide (20nM), TMAO or TMAO+Triptolide for 24hrs. Scale bar=25μm.

2). An anti-inflammatory and neuroprotective biomimetic nanoplatform for repairing spinal cord injury. Bioactive Materials, 2022 (PubMed: 35845318) [IF=18.9]

Application: WB    Species: Mice    Sample: spinal cord

Fig. 2 Macrophage phenotype regulation based on RA@BSA@Cur NPs. (a) The schematic illustration of RA@BSA@Cur NPs regulated macrophage polarization under LPS. (b) The immunofluorescence and quantification results of RAW264.7 cultured different BSA-related NPs in LPS condition. CD86+ M1 macrophage (yellow arrow) and CD206+ M2 macrophage (white arrow), nuclei (DAPI: blue). Scale bar, 50 μm. (c&d) The flow cytometry analysis and quantification results of RAW264.7 (gated on F4/80+) cultured different BSA-related NPs in LPS condition. (e) The macrophages' M1 inhibition and M2 promotion regulated by RA@BSA@Cur NPs may be through the NF-κB pathway. (f) The inflammatory factors IL-6, TNF-α, and IL-4 changes after RA@BSA@Cur treatment. (n = 3 independent samples). Statistical differences were determined by using the Analysis of Variance (ANOVA) with Bonferroni's multiple comparison test (*p < 0.05, **p < 0.01, ***p < 0.001, ns: no significant; a.u. means arbitrary unit).

3). Tetrahedral Framework Nucleic Acids Based Small Interfering RNA Targeting Receptor for Advanced Glycation End Products for Diabetic Complications Treatment. ACS Nano, 2023 (PubMed: 37751401) [IF=17.1]

4). Gut dysbiosis promotes prostate cancer progression and docetaxel resistance via activating NF-κB-IL6-STAT3 axis. Microbiome, 2022 (PubMed: 35710492) [IF=15.5]

Application: WB    Species: Mouse    Sample: tumor tissue

Fig. 3 Intratumoral LPS activated NF-κB-IL6-STAT3 axis. A LPS levels in mouse feces and serum by ELISA; HE staining (scale bar, 50 μm) and histology score for colon tissue in the Abx and NC group. B Immunohistochemistry (scale bar, 50 μm) for LPS in subcutaneous and orthotopic tumor tissues and western blot of intratumoral LPS levels from three biological duplications for the Abx and NC group. C Transcription levels of cytokines by RT-qPCR and protein levels of IL6 in cell supernatant by ELISA in RM-1 cultured with LPS (100 μg/ml) for 24 h. D, E Immunofluorescence (scale bar, 100 μm) for p-p65 and p-STAT3 in RM-1 cultured with or without LPS for 24 h; western blot of relative proteins for RM-1 cultured with LPS at different concentrations for 24 h; transcription levels of IL6 by RT-qPCR and western blot of relative proteins in RM-1 cultured with LPS or LPS with BAY-11-7082 for 24 h; protein levels of p-STAT3 and STAT3 in RM-1 cultured with CM or CM with antibody-IL6 for 24 h. F, G IL6 levels in tumor tissue lysate and serum by ELISA. Western blot of relative proteins in tumor from three biological duplications. Immunohistochemistry of tumor tissues for p-p65- and p-STAT3-positive cell (scale bar, 50 μm). Statistical significance was assessed by unpaired Student’s T-test or LSD in one-way ANOVA. *p < 0.05, **p < 0.01, and ***p < 0.001: compared to the NC group; #p < 0.05, ##p < 0.01, and ###p < 0.001: compared to the LPS or CM group

Application: IF/ICC    Species: Mouse    Sample: tumor tissue

Fig. 3 Intratumoral LPS activated NF-κB-IL6-STAT3 axis. A LPS levels in mouse feces and serum by ELISA; HE staining (scale bar, 50 μm) and histology score for colon tissue in the Abx and NC group. B Immunohistochemistry (scale bar, 50 μm) for LPS in subcutaneous and orthotopic tumor tissues and western blot of intratumoral LPS levels from three biological duplications for the Abx and NC group. C Transcription levels of cytokines by RT-qPCR and protein levels of IL6 in cell supernatant by ELISA in RM-1 cultured with LPS (100 μg/ml) for 24 h. D, E Immunofluorescence (scale bar, 100 μm) for p-p65 and p-STAT3 in RM-1 cultured with or without LPS for 24 h; western blot of relative proteins for RM-1 cultured with LPS at different concentrations for 24 h; transcription levels of IL6 by RT-qPCR and western blot of relative proteins in RM-1 cultured with LPS or LPS with BAY-11-7082 for 24 h; protein levels of p-STAT3 and STAT3 in RM-1 cultured with CM or CM with antibody-IL6 for 24 h. F, G IL6 levels in tumor tissue lysate and serum by ELISA. Western blot of relative proteins in tumor from three biological duplications. Immunohistochemistry of tumor tissues for p-p65- and p-STAT3-positive cell (scale bar, 50 μm). Statistical significance was assessed by unpaired Student’s T-test or LSD in one-way ANOVA. *p < 0.05, **p < 0.01, and ***p < 0.001: compared to the NC group; #p < 0.05, ##p < 0.01, and ###p < 0.001: compared to the LPS or CM group

Application: IHC    Species: Mouse    Sample: tumor tissue

Fig. 3 Intratumoral LPS activated NF-κB-IL6-STAT3 axis. A LPS levels in mouse feces and serum by ELISA; HE staining (scale bar, 50 μm) and histology score for colon tissue in the Abx and NC group. B Immunohistochemistry (scale bar, 50 μm) for LPS in subcutaneous and orthotopic tumor tissues and western blot of intratumoral LPS levels from three biological duplications for the Abx and NC group. C Transcription levels of cytokines by RT-qPCR and protein levels of IL6 in cell supernatant by ELISA in RM-1 cultured with LPS (100 μg/ml) for 24 h. D, E Immunofluorescence (scale bar, 100 μm) for p-p65 and p-STAT3 in RM-1 cultured with or without LPS for 24 h; western blot of relative proteins for RM-1 cultured with LPS at different concentrations for 24 h; transcription levels of IL6 by RT-qPCR and western blot of relative proteins in RM-1 cultured with LPS or LPS with BAY-11-7082 for 24 h; protein levels of p-STAT3 and STAT3 in RM-1 cultured with CM or CM with antibody-IL6 for 24 h. F, G IL6 levels in tumor tissue lysate and serum by ELISA. Western blot of relative proteins in tumor from three biological duplications. Immunohistochemistry of tumor tissues for p-p65- and p-STAT3-positive cell (scale bar, 50 μm). Statistical significance was assessed by unpaired Student’s T-test or LSD in one-way ANOVA. *p < 0.05, **p < 0.01, and ***p < 0.001: compared to the NC group; #p < 0.05, ##p < 0.01, and ###p < 0.001: compared to the LPS or CM group

5). CPAL, as a New Mediator of Cardiomyocyte Metabolic Alterations and Pyroptosis, Regulates Myocardial Infarction Injury in Mice. Engineering, 2023 [IF=12.8]

Application: WB    Species: Mouse    Sample:

Fig. 7. CPAL is an upstream regulator of NFκB. (a) Two specific regions (76–127 nt and 209–260 nt) of CPAL that were found to bind with NFκB using the online prediction website. (b) RIP analysis for CPAL-NFκB interaction. Immunoprecipitation of NFκB acquired a large amount of CPAL (**P  < 0.01, compared with anti-IgG; n  = 3). (c) Expression of P-NFκB and NFκB at the protein levels in each group (**P  < 0.01, compared with sham group; ##P  < 0.01, compared with +AAV9-sh-NC group; n  = 4 to n  = 6). (d) Immunohistochemical staining of P-NFκB protein in the infarct border zones of the left ventricular (**P  < 0.01, compared with sham group; ##P  < 0.01, compared with +AAV9-sh-NC group; n  = 3 to n  = 4). (e) Expression of P-NFκB and NFκB at the protein level in cardiomyocytes of control, LPS, +si-NC, and +si-CPAL groups (**P  < 0.01, compared with control; #P < 0.05, compared with LPS group; n  = 9). (f) Staining for P-NFκB (green) and α-actinin (red) proteins in the cardiomyocytes of control, LPS, +si-NC, and +si-CPAL groups (blue: DAPI; n  = 4). (g) P-NFκB expression in the neonatal mouse cardiomyocytes of pcDNA3.1-OE-NC, pcDNA3.1-OE-CPAL, +ODNs-CPAL, and +ODNs-NC groups (**P  < 0.01, compared with pcDNA3.1-OE-NC; ##P  < 0.01, compared with pcDNA3.1-OE-CPAL; n  = 5).

6). Mitochondrial STAT3 exacerbates LPS-induced sepsis by driving CPT1a-mediated fatty acid oxidation. Theranostics, 2022 (PubMed: 34976224) [IF=12.4]

Application: WB    Species: Mice    Sample: Lung tissues

Figure 8 Curcumin relieved LPS-induced sepsis. (A) After the last 4-OHT injection, Rosa26LSL-MLS-Stat3 and Rosa26LSL-MLS-Stat3;UbcERT2Cre/+ mice were injected with curcumin prior to LPS. Survival was monitored every 6 - 12 h after injection. The data shown represent a combination of two independent experiments. Statistical comparison of survival was performed with a log-rank test. **, P < 0.01, *, P < 0.05. (B) Serum samples were obtained from these mice and subjected to ELISA to examine MCP-5 levels. (C-D) The serum levels of LDH and albumin were assessed. (E) Lung tissues were removed, and the percentages of macrophages were analyzed by flow cytometry. (B-E) The data are representative of one of two independent experiments. Significance was calculated using a two-way ANOVA. **, P < 0.01, * P < 0.05, n.s., not significant. (F) Rosa26LSL-MLS-Stat3 and Rosa26LSL-MLS-Stat3;LyzCre/+ mice were injected with 10 mg/kg curcumin prior to LPS (20 mg/kg). Survival was monitored every 6 - 12 h after injection. Statistical comparison of survival was performed with a log-rank test. **, P < 0.01, *, P < 0.05. (G) BMDMs of the indicated genotypes were treated with 8 μM curcumin for 20 h and incubated with either 100 ng/mL LPS (30 min) or 100 ng/mL IFN-α (1 h) before harvesting. The cells were harvested, lysed, and subjected to a Western blot analysis. The membranes were blotted with the indicated antibodies. (H) Cells were treated with 8 μM curcumin for 20 h and incubated with 100 ng/mL LPS for another 15 min. Nuclei and cytoplasmic fractions were extracted. The levels of the indicated proteins were analyzed by a Western blot analysis. (I) BMDMs were treated with curcumin. After 20 h, these cells were stimulated with or without 100 ng/mL LPS for 6 h before loading. BMDMs were then treated sequentially with BSA-conjugated palmitate, oligomycin, FCCP, and ETO as indicated. The data are representative of one of two independent experiments performed with n = 5. (J) 4-OHT-treated BMDMs were treated with curcumin for 20 h, and then, the BMDMs were exposed to 100 ng/mL LPS and 10 μmol/L MG132 for an additional 6 h before harvesting. The cell lysates were isolated, IP with an anti-CPT1a antibody and analyzed by a Western blot analysis using an anti-ubiquitin antibody. (G, H and J) The results shown are representative of one of three independent experiments. (K) Schematic representation of the mitochondrial STAT3-mediated increase in FAO in a USP50-dependent manner in the LPS-induced sepsis model. Upon the LPS stimulation, mitochondrial STAT3 could induce CPT1a stabilization by promoting USP50 expression and facilitating CPT1a-USP50 binding, leading to a switch from glucose to an increased reliance on FAO for ATP production, which, in turn, further enhanced NF-κB nuclear localization, leading to a great amount of cytokine production.

7). Molecular mechanism of interleukin-17A regulating airway epithelial cell ferroptosis based on allergic asthma airway inflammation. Redox biology, 2023 (PubMed: 38035662) [IF=11.4]

8). Arsenic retention in erythrocytes and excessive erythrophagocytosis is related to low selenium status by impaired redox homeostasis. Redox Biology, 2022 (PubMed: 35500533) [IF=11.4]

Application: WB    Species: Mice    Sample: splenic macrophages

Fig. 6 Inflammation in splenic macrophages that phagocytosed As-exposed erythrocytes. (A–B) Relative mRNA levels of targets genes in macrophages that phagocytosed control erythrocytes or As-exposed erythrocytes were determined by qPCR (n = 3 per group) and data are presented. (C) Protein levels of targets in splenic macrophages phagocytosed control erythrocytes or As-exposed erythrocytes were analyzed by western blotting. (D) The levels of p-NFκB, iNOS and Cox-2 in mouse spleen sections were analyzed by immunohistochemistry. Differences between groups were analyzed by using the unpaired Student’s t tests. ***p < 0.001, **p < 0.005 or *p < 0.05 indicates significant difference.

Application: IHC    Species: Mice    Sample: splenic macrophages

Fig. 6 Inflammation in splenic macrophages that phagocytosed As-exposed erythrocytes. (A–B) Relative mRNA levels of targets genes in macrophages that phagocytosed control erythrocytes or As-exposed erythrocytes were determined by qPCR (n = 3 per group) and data are presented. (C) Protein levels of targets in splenic macrophages phagocytosed control erythrocytes or As-exposed erythrocytes were analyzed by western blotting. (D) The levels of p-NFκB, iNOS and Cox-2 in mouse spleen sections were analyzed by immunohistochemistry. Differences between groups were analyzed by using the unpaired Student’s t tests. ***p < 0.001, **p < 0.005 or *p < 0.05 indicates significant difference.

9). LncRNA AK023391 promotes tumorigenesis and invasion of gastric cancer through activation of the PI3K/Akt signaling pathway. Journal of Experimental & Clinical Cancer Research, 2017 (PubMed: 29282102) [IF=11.3]

Application: WB    Species: human    Sample: Gastric cancer cells

Fig. 8 | LncRNA AK023391 was involved in the regulation of the PI3K/Akt signaling pathway.e-f Western blotting validation of the effects of AK023391 knockdown on the expression of PI3K/Akt, NF-κB, p53, and FOXO3a pathways, and their downstream transcription factors c-myb, cyclinB1/G2, and BCL-6 in HGC 27, AGS, and SGC-7901 cells

Application: IHC    Species: human    Sample: xenograft tumor

Fig. 9 | Knockdown of AK023391 inhibited tumor growth in vivo. d IHC analysis of the expression levels of Ki-67, p-FOXO3a, p-PI3K, p-Akt, and p-NF-κB in xenograft tumor tissues treated with si-AK023391 and in the NC group (original magnification, ×200).

10). Water-extracted Lonicera japonica polysaccharide attenuates allergic rhinitis by regulating NLRP3-IL-17 signaling axis. Carbohydrate Polymers, 2022 (PubMed: 36184153) [IF=11.2]

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