Nox4 Antibody

Western Blot: Nox4 Antibody [NB110-58851] - Whole cell protein from human HeLa, Hek293, HepG2, mouse Neuro2A and rat PC12 cells was separated on a 7.5% gel by SDS-PAGE, transferred to PVDF membrane and blocked in 5% BSA in TBST. The membrane was probed with 2.0 ug/ml anti-Nox4 in 1% BSA and detected with an anti-rabbit HRP secondary antibody using chemiluminescence. Nox4 is detected at approx. 70 kDa (arrowhead)

Immunocytochemistry/Immunofluorescence: Nox4 Antibody [NB110-58851] - HeLa cells were fixed in 10% buffered formalin for 10 min and permeabilized in 0.1% Triton X-100 in PBS for 10 min. Cells were incubated with antibodies to Nox4 (NB110-58851) and the ER marker Calreticulin (NBP1-47518), each at 20 ug/ml for 1 hour at room temperature. The coverslips were washed 3x in PBS and incubated with Alexa-Fluor 488 anti-rabbit secondary antibody. (green) and DyLight 550 anti-mouse secondary antibody. The merged image shows the co-localization of Nox4 and Calreticulin in the ER.

Immunohistochemistry: Nox4 Antibody [NB110-58851] - The expression of Nox4, p21, and Rb was upregulated in degenerative discs. Immunohistochemical staining for Nox4 in NP specimens. Image collected and cropped by CiteAb from the following publication (//www.hindawi.com/journals/omcl/2017/7426458/) licensed under a CC-BY licence.

Immunohistochemistry: Nox4 Antibody [NB110-58851] - Analysis using the Biotin conjugate of NB110-58851. Staining of NOX4 in proximal convoluted tubules of human kidney.

Immunocytochemistry/Immunofluorescence: Nox4 Antibody [NB110-58851] - HeLa cells were fixed in 10% buffered formalin for 10 min and permeabilized in 0.1% triton X-100 in PBS for 10 min. Cells were incubated with NB110-58851 at 20 ug/ml for 1 hour at room temperature, washed 3x in PBS and incubated with Alexa-Fluor 488 anti-rabbit secondary antibody. Nox4 (Green) was detected in the ER.

Western Blot: Nox4 Antibody [NB110-58851] - Analysis using the Biotin conjugate of NB110-58851. Detection of NOX4 in human kidney lysates uisng NB110-58851 at 0.5 ug/ml. Band observed at 31 kDa may represent reported splice isoform.

• Reactivity: Hu, Mu, Rt, Po, Pm
• Applications: WB, Flow, ICC/IF, IHC, IHC-P, IP
• Clonality: Polyclonal
• Host: Rabbit
• Conjugate: Unconjugated
• Concentration: 1.0 mg/ml

Nox4 Antibody Summary

• Immunogen: A synthetic peptide made to a C-terminal region (within residues 500-578) of the human NOX4 protein sequence. [Swiss-Prot# Q9NPH5].
• Localization: Endoplasmic reticulum, endoplasmic reticulum membrane, multi-pass membrane protein, cell membrane, nucleus (probable).
• Isotype: IgG
• Clonality: Polyclonal
• Host: Rabbit
• Gene: NOX4
• Purity: Immunogen affinity purified

Applications/Dilutions

• Dilutions:
  • Western Blot 2 - 4 ug/ml
  • Flow Cytometry
  • Immunocytochemistry/Immunofluorescence 10 - 20 ug/ml
  • Immunohistochemistry 10 - 20 ug/ml
  • Immunohistochemistry-Paraffin 10 - 20 ug/ml
  • Immunoprecipitation
• Application Notes: This NOX4 antibody is useful for Western blot, ICC and Immunohistochemistry paraffin embedded sections. In Western Blot this NOX4 antibody recognizes bands at ~70kDa. Use in immunprecipitation has reported in scientific literature (PMID 25062272)
• Control:

  Human Kidney Whole Tissue Lysate (Adult Whole Normal)

Reactivity Notes

Human, mouse, rat, pig and primate.

Packaging, Storage & Formulations

• Storage: Store at 4C short term. Aliquot and store at -20C long term. Avoid freeze-thaw cycles.
• Buffer: PBS (pH 7.4)
• Preservative: 0.02% Sodium Azide
• Concentration: 1.0 mg/ml
• Purity: Immunogen affinity purified

Alternate Names for Nox4 Antibody

• EC 1.6.3
• Kidney oxidase-1
• Kidney superoxide-producing NADPH oxidase
• KOX
• KOX-1
• KOX-1Renal NAD(P)H-oxidase
• NADPH oxidase 4
• Nox4
• RENOX
• RENOXEC 1.6.3.-

Background

NOX4 (NADPH oxidase 4) is a constitutive NADPH oxidase which forms a complex with CYBA/p22phox generates superoxide intracellularly, and is found primarily in vascular cells, fibroblasts, osteoclasts and is abundantly expressed in the kidney. NOX4 regulates signaling cascades through phosphatase inhibition, functions in oxygen sensor regulation in the KCNK3/TASK-1 potassium channel, and also demonstrates HIF1 alpha activity. NOX4 has also been implicated in regulation of insulin signaling cascade, apoptosis, bone resorption and LPS-mediated activation of NFkB. In vascular smooth muscle cells, NOX4 is positively regulated by Poldip2 which is a Nox4/p22phox interacting protein and Nox4/p22phox-Poldip2 complex regulates Rho-dependent cytoskeletal reorganization as well as cellular migration. TGF-beta is another positive regulator of Nox4 which is important in vascular fibrosis and differentiation. Unlike Nox1/Nox2, Nox4 is constitutively active, producing primarily H2O2 rather than O2. Nox4 contributes to basal ROS production through its constitutive activity and to increased ROS generation when stimulated by Ang II, glucose, TNF alpha and growth factors. NOX4 has been implicated in hypertension, atherosclerosis, cardiovascular and renal complications of diabetes, and in remodeling of pulmonary arteries in hypertension.

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

Publications for Nox4 Antibody (NB110-58851)(43)

Publications using NB110-58851

• Feng C, Zhang Y, Yang M et al. Oxygen-Sensing Nox4 Generates Genotoxic ROS to Induce Premature Senescence of Nucleus Pulposus Cells through MAPK and NF-kappa B Pathways Oxid Med Cell Longev. 2017 Sep 23 [PMID: 29147462] (WB, Rat)
• Miranda-Alves, L. Influence of Organotin on Thyroid Morphophysiological Status JOURNAL OF ENVIRONMENT AND HEALTH SCIENCE Jul 17 2015 (WB, Rat)
• Garcia-Arroyo FE, Gonzaga G, Munoz-Jimenez I et al. Antioxidant supplements as a novel mean for blocking recurrent heat stress-induced kidney damage following rehydration with fructose-containing beverages Free Radic. Biol. Med. Jun 15 2019 [PMID: 31212064] (WB, Rat)
• Stuart JA, Fonseca J, Moradi F et al. How Supraphysiological Oxygen Levels in Standard Cell Culture Affect Oxygen-Consuming Reactions. Oxid Med Cell Longev. Sep 30 2018 [PMID: 30363917] (WB, Human, Mouse)
• Varga ZV, Pipicz M, Baan JA et al. Alternative Splicing of NOX4 in the Failing Human Heart. Front Physiol 2017 Nov 22 [PMID: 29204124] (WB, Human)
• Bidwell PA, Liu GS, Nagarajan N et al. HAX-1 regulates SERCA2a oxidation and degradation J. Mol. Cell. Cardiol. 2017 Nov 20 [PMID: 29169992] (WB)
• Goitre L, DiStefano PV, Moglia A et al. Up-regulation of NADPH oxidase-mediated redox signaling contributes to the loss of barrier function in KRIT1 deficient endothelium Sci Rep 2017 Aug 15 [PMID: 28811547] (IP, Mouse)
• Feng C, Zhang Y, Yang M et al. Oxygen-sensing Nox4 generates genotoxic ROS to induce premature senescence of downloads.hindawi.com 2017 Aug 08 (WB, Rat)
• Shi YW, Zhang XC, Chen C et al. Schisantherin A attenuates ischemia/reperfusion-induced neuronal injury in rats via regulation of TLR4 and C5aR1 signaling pathways Brain Behav. Immun. 2017 Jul 08 [PMID: 28690033] (Rat)
• Fierro-Fernandez M, Busnadiego O, Sandoval P et al. miR-9-5p suppresses pro-fibrogenic transformation of fibroblasts and prevents organ fibrosis by targeting NOX4 and TGFBR2. EMBO Rep 2015 Oct [PMID: 26315535] (WB)
• Stanicka J, Russell EG, Woolley JF, Cotter TG. NADPH oxidase-generated hydrogen peroxide induces DNA damage in mutant FLT3-expressing leukemia cells J. Biol. Chem. 2015 Feb 19 [PMID: 25697362] (WB, ICC/IF, Human, Mouse)
• Nisimoto Y, Diebold BA, Constentino-Gomes D, Lambeth JD. Nox4: A Hydrogen Peroxide-Generating Oxygen Sensor. Biochemistry 2014 Aug 01 [PMID: 25062272] (IP, WB, Human)
  
  Details:
  NOX4 antibody used for WB and IP in Nox4 -transfected HEK293 cells (Supplemental Figures 3 and 4).
• de Figueiredo SP, Neto RAL., et al. Administration of 3,5-diiodothyronine (3,5-T2) causes central hypothyroidism and stimulates thyroid-sensitive tissues. J Endocrinol 2014 Apr 01 [PMID: 24692290] (WB, Rat)
• Ogrunc M, Di Micco R, Liontos M et al. Oncogene-induced reactive oxygen species fuel hyperproliferation and DNA damage response activation. Cell Death Differ. 2014 Mar 03 [PMID: 24583638] (IHC-P, ICC/IF, Mouse, Human)
• Gurbuz I, Ferralli J, Roloff T et al. SAP domain-dependent Mkl1 signaling stimulates proliferation and cell migration by induction of a distinct gene set indicative of poor prognosis in breast cancer patients. Mol. Cancer 2014 Feb 25 [PMID: 24495796] (WB, Mouse)
  
  Details:
  NOX4 antibody used for WB on whole-cell extracts from the three HC11 strains overexpressing either FL-, mutB1- or delta-SAP-Mkl1 constructs (Figure S2).
• Dunlop K, Gosal K, Kantores C et al. Therapeutic hypercapnia prevents inhaled nitric oxide-induced right-ventricular systolic dysfunction in juvenile rats. Free Radic. Biol. Med. 2014 Jan 11 [PMID: 24423485] (Rat)
• Feng W, Chumley P, Allon M et al. The Transcription Factor E26 Transformation-Specific Sequence-1 Mediates Neointima Formation in Arteriovenous Fistula. J Am Soc Nephrol. 2013 Nov 7 [PMID: 24203999]
• Khazim K, Gorin Y, Cavaglieri RC et al. The antioxidant silybin prevents high glucose-induced oxidative stress and podocyte injury in vitro and in vivo. Am J Physiol Renal Physiol 2013 Sep 1 [PMID: 23804455]
• Varga ZV, Kupai K, Szucs G et al. MicroRNA-25-dependent up-regulation of NADPH oxidase 4 (NOX4) mediates hypercholesterolemia-induced oxidative/nitrative stress and subsequent dysfunction in the heart. J Mol Cell Cardiol 2013 May 27 [PMID: 23722270] (IHC-P, WB, Rat)
• Zhang B1, Liu Z, Hu X. Zhang B, Liu Z, Hu X. Inhibiting cancer metastasis via targeting NAPDH oxidase 4. Biochem Pharmacol 2013 May 17 [PMID: 23688500] (IHC, Mouse)
  
  Details:
  This publication used the HRP conjugated form of this antibody (NB110-58851H)
• Dick AS, Ivanovska J, Kantores C et al. Cyclic stretch stimulates nitric oxide synthase-1-dependent peroxynitrite formation by neonatal rat pulmonary artery smooth muscle. Free Radic Biol Med 2013 Apr 22 [PMID: 23619128] (WB, Rat)
• Santos MC, Louzada RA, Souza EC et al. Diabetes mellitus increases reactive oxygen species production in the thyroid of male rats. Endocrinology 2013 Mar [PMID: 23407453]
• Shah A, Xia L, Goldberg H et al. Thioredoxin-interacting protein mediates high glucose-induced reactive oxygen species (ROS) generation by mitochondria and the NADPH oxidase, Nox4, in mesangial cells. J Biol Chem 2013 Jan 17 [PMID: 23329835] (WB, Mouse)
• Sato-Horiguchi C, Ogawa D, Wada J et al. Telmisartan Attenuates Diabetic Nephropathy by Suppressing Oxidative Stress in db/db Mice. Nephron Exp Nephrol 2013 Jan 10 [PMID: 23307263]
• Ilatovskaya DV, Pavlov TS, Levchenko V, Staruschenko A. ROS production as a common mechanism of ENaC regulation by EGF, insulin and IGF-1 Am J Physiol Cell Physiol 2012 Nov 7 [PMID: 23135700] (WB, Mouse)
• Yan Q, Gao K, Chi Y et al. NADPH oxidase-mediated upregulation of connexin43 contributes to podocyte injury Free Radic Biol Med 2012 Jul 20 [PMID: 22824863] (WB, Mouse)
• Ju-Young Moon. Angiotensin II-Induced Mitochondrial Nox4 Is a Major Endogenous Source of Oxidative Stress in Kidney Tubular Cells plos One 2012 [PMID: 22808054] (WB, ICC/IF, Rat)
• Siuda D, Zechner U, El Hajj N et al. Transcriptional regulation of Nox4 by histone deacetylases in human endothelial cells Basic Res Cardiol 2012 Sep [PMID: 22791246] (WB, Human)
• Jiang JX, Chen X, Serizawa N, Szyndralewicz C, Page P, Schroder K, Brandes RP, Devaraj S, Torok NJ. Liver fibrosis and hepatocyte apoptosis are attenuated by GKT137831, a novel NOX4/NOX1 inhibitor in vivo. Free Radic Biol Med. 2012 May 19. [PMID: 22618020] (IHC, ICC/IF, WB, Human, Mouse)
• Rezonzew G, Chumley P, Feng W et al. Nicotine exposure and the progression of chronic kidney disease: role of the alpha 7-nicotinic acetylcholine receptor Am J Physiol Renal Physiol 2012 Jul [PMID: 22552933] (WB, ICC/IF, Rat)
• Xie W, Parker JL, Heaps CL. Effect of exercise training on nitric oxide and superoxide/H2O2 signaling pathways in collateral-dependent Porcine coronary arterioles. J Appl Physiol;112(9):1546-55. 2012 May. [PMID: 22323648] (WB, Porcine)
• Chen J, Chen JK, Harris RC. Angiotensin II Induces Epithelial-to-Mesenchymal Transition in Renal Epithelial Cells through Reactive Oxygen Species/Src/Caveolin-Mediated Activation of an Epidermal Growth Factor Receptor-Extracellular Signal-Regulated Kinase Signaling Pathway. Mol Cell Biol;32(5):929-40. 2012 Mar. [PMID: 22215616]
• Goodson P, Kumar A, Jain L, Kundu K, Murthy N, Koval M, Helms MN. Nadph oxidase regulates alveolar epithelial sodium channel activity and lung fluid balance in vivo via OFormula signaling. Am J Physiol Lung Cell Mol Physiol;302(4):L410-9. 2012 Feb. [PMID: 22160304]
• Bruce-Keller AJ, Gupta S, Knight AG et al. Cognitive impairment in humanized APPxPS1 mice is linked to A-beta (1-42) and NOX activation. Neurobiol Dis. Dec. 2011 [PMID: 21798347] (WB, Mouse)
• Clavreul N, Sansilvestri-Morel P, Magard D et al. (Pro)renin promotes fibrosis gene expression in HEK cells through a Nox4-dependent mechanism. Am J Physiol Renal Physiol. Jun. 2011 [PMID: 21411480] (WB, Human)
• Carnesecchi S, Deffert C, Donati Y et al. A key role for NOX4 in epithelial cell death during development of lung fibrosis. Antioxid Redox Signal. 2011 Mar 11. [PMID: 21391892]
• Kodera R, Shikata K, Kataoka HU et al. Glucagon-like peptide-1 receptor agonist ameliorates renal injury through its anti-inflammatory action without lowering blood glucose level in a rat model of type 1 diabetes. Diabetologia. Apr. 2011 [PMID: 21253697] (IHC-P, ICC/IF, Rat)
• Handayaningsih AE, Iguchi G, Fukuoka H et al. Reactive oxygen species play an essential role in IGF-I signaling and IGF-I-induced myocyte hypertrophy in C2C12 myocytes. Endocrinology 2011 Mar [PMID: 21239445]
• Wang R, Dashwood WM, Nian H et al. NADPH oxidase overexpression in human colon cancers and rat colon tumors induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) Int J Cancer 2011 Jun 1 [PMID: 20715105] (WB, IHC, Rat, Human)
• Boudreau HE, Emerson SU, Korzeniowska A et al. Hepatitis C virus (HCV) proteins induce NADPH oxidase 4 expression in a transforming growth factor beta-dependent manner: a new contributor to HCV-induced oxidative stress. J Virol;83(24):12934-46. 2009 Dec. [PMID: 19812163] (WB, Human)
• Weyemi U, Caillou B, Talbot M et al. Intracellular expression of reactive oxygen species-generating NADPH oxidase NOX4 in normal and cancer thyroid tissues. Endocr Relat Cancer;17(1):27-37. 2010 Jan 29. [PMID: 19779036] (WB, IHC-P, Human)
• Kim J, Park J-W, Park KM. Increased superoxide formation induced by irradiation preconditioning triggers kidney resistance to ischemia-reperfusion injury in mice. Am J Physiol Renal Physiol;296(5):F1202-1211. 2009 [PMID: 19193722] (WB, Mouse)
• Basuroy S, Bhattacharya S, Leffler CW et al. Nox4 NADPH oxidase mediates oxidative stress apoptosis caused by TNF-{alpha} in cerebral vascular endothelial cells. Am J Physiol Cell Physiol;296(3):C422-432. 2009 [PMID: 19118162] (WB, Porcine)

Reviews for Nox4 Antibody (NB110-58851) (2)

Average Rating: 3.5

(Based on 2 reviews)

We have 2 reviews tested in 2 species: Mouse, Rat.

reviewed by: Netanya Spencer WB Mouse 10/05/2011

Summary

• Application: Western Blot
• Sample Tested: murine liver, Sample Amount: 100 ug
• Species: Mouse
• Lot: E
• Comments: Unlike the image on the website, I got at least 6 bands at different molecular weights. I don't know if they are all real or not. I didn't have a good positive or negative control so I don't know which, if any, bands were the right protein. There may be splice isoforms and/or posttranslational modifications.

Blocking

• Blocking Details: Blocking Buffer: 5% milk 1% BSA in TBS, Blocking Time: 1 hour, Blocking Temp: Room temperature

Primary Anitbody

• Dilution Ratio: Primary Ab Dilution Ratio: 1:300, Incubation Dilution Buffer: 5% milk 1% BSA in TBS, Incubation Time: ON-4C then 2.5hrs-RT

Secondary Antibody

• Secondary Description: Secondary Ab: HRP-Goat Anti-Rabbit IgG(H+l), Secondary Ab Dilution Ratio: 1:2000
• Secondary Manufacturer Cat#: 00001-2 ProteinTech Group, Inc

Details

• Detection Notes: Detection Method: ECL on Chem-Doc-It, Exposure Time: 1-10 min, Bands: not sure, ranging from 30-200 Kda

Comments

• Comments: Unlike the image on the website, I got at least 6 bands at different molecular weights. I don't know if they are all real or not. I didn't have a good positive or negative control so I don't know which, if any, bands were the right protein. There may be splice isoforms and/or posttranslational modifications.

reviewed by: Anonymous WB Rat 03/26/2009

Summary

• Application: Western Blot
• Sample Tested: rat liver whole homogenate/primary hepatocyte cell lysate, Sample Amount: 45
• Species: Rat
• Lot: C1
• Comments: Really pretty happy after my experiences with other Nox isoform antibodies. Will be running the positive control peptide soon.

Blocking

• Blocking Details: Blocking Buffer: 5% NFDM, Blocking Time: overnight, Blocking Temp: 4 degrees Celsius

Primary Anitbody

• Dilution Ratio: Primary Ab Dilution Ratio: 1/1000, Incubation Dilution Buffer: TBST, Incubation Time: overnight, Incubation Temp: 4F

Secondary Antibody

• Secondary Description: Secondary Ab Dilution Ratio: 1/20000

Details

• Detection Notes: Method:ECL-SuperSignal Dura BioRad ChemiDoc, Exposure Time:90-120sec, Specific Bands:31, 67kDa, Non-Specific Bands:45, 50, 90kDa

Comments

• Comments: Really pretty happy after my experiences with other Nox isoform antibodies. Will be running the positive control peptide soon.

FAQs for Nox4 Antibody (NB110-58851). (Showing 1 - 3 of 3 FAQs).

1. I want to know the cell fixation condition when rabbit anti-Nox4 antibodies (cat, no. NB110-58851)are used for immunohistochemistry. Is that Parafolmaldehyde or MethanoL? What concentration, How long? You showed nice IHC stain of Nox4 in Kidney in the data sheet. 1.Samples are fixed by formaldehyde? 2.What is the method of retrieval of samples before staining. For example, It was done by treatment with EDTA or citrate ?
   • I am delighted to hear you are pleased with our image! The fixation method used is Paraffin embedded with a dilution of 10-20 ug/ml. Please see our general protocol. This antibody has been tested by one of our innovators so we do not have their protocol. I would suggest fixing in 2% paraformaldehyde over night and citrate antigen retrieval as this is the most common form of antigen retrieval. If you face any problems through out your staining we are more than happy to help and can suggest alternative methods to gain a good level of staining.
2. I would like to know the immunogen peptide sequence for Nox4 (NB110 -58851).
   • Unfortunately the peptide sequence for this antibody is deemed proprietary and cannot be provided. However, we can tell you that it is a synthetic peptide made to a C-terminal region (within residues 500-578) of the human NOX4 protein sequence [Swiss-Prot# Q9NPH5]. Most likely this peptide will be approx. 10-20 amino acids long within that range.
3. Do you have an alternative product for NOX4 Antibody (NB110-58851). I will be using it for porcine.
   • NB110-58849 is validated in the same species (apart from porcine) and applications as NB110-58851 and should be a suitable alternative for you. NB110-58851 was raised to a synthetic peptide made to a C-terminal region (within residues 500-578) of the human NOX4 protein, whilst NB110-58849 was raised to a synthetic peptide made to an internal region (between residues 100-200) of the human NOX4 protein. Both antibodies are rabbit polyclonals. As NB110-58849 has not yet been tested in porcine samples, you may be interested in our Innovator's Reward programme. This allows you to try our primary antibodies in an untested species or application, without the financial risk of failure. To participate you simply need to go to the antibody's webpage and complete an online review with an image, detailing the positive or negative results of your study. In return you will receive a discount voucher for 100% of the purchase price of the reviewed product. More details of this programme can be found here: Innovator's Reward Program. I ran an alignment for you between the human NOX4 sequence and an unreviewed porcine NOX4 sequence from Uniprot (accession number F1STQ7.). The homology is high between the two proteins. Please do, however, be aware that this porcine sequence is unreviewed.

Bioinformatics

• Gene Symbol: NOX4