HIF-2 alpha/EPAS1 Antibody Pack

Immunohistochemistry: HIF-2 alpha/EPAS1 Antibody Pack [NB100-902HIF2] - Analysis of HIF-2 alpha in human endometrium using. Donkey anti-mouse Alexa Fluor 488 secondary antibody was used. Image from verified customer review. HIF-2 alpha/EPAS1 Antibody (ep190b) [NB100-132]

Western Blot: HIF-2 alpha/EPAS1 Antibody Pack [NB100-902HIF2] - WNT11 is induced by hypoxia or hypoxic mimetics in different cell types. Immunoblot analyses of HeLa cells under normal air or hypoxia for 24 hrs. Image collected and cropped by CiteAb from the following publication (//www.nature.com/articles/srep21520) licensed under a CC-BY licence. HIF-2 alpha/EPAS1 Antibody [NB100-122]

Simple Western: HIF-2 alpha/EPAS1 Antibody Pack [NB100-902HIF2] - Lane view shows a specific band for HIF-2 alpha in 0.5 mg/ml of Hypoxic HeLa lysate. This experiment was performed under reducing conditions using the 12-230 kDa separation system. HIF-2 alpha/EPAS1 Antibody (ep190b) [NB100-132]

Knockout Validated: HIF-2 alpha/EPAS1 Antibody Pack [NB100-902HIF2] - HIF-1alpha is the predominant transcriptional regulator of WNT11 expression during hypoxia. EMSCs isolated from the indicated mouse genotypes were infected with lentivirus expressing GFP or Cre recombinase. Non-infected cells and GFP infected cells served as controls. Immunoblot analyses of EMSCs derived from the indicated genotypes treated with 0.1 mM DMOG for 24 hrs. Attenuated WNT11 expression in Hif-1alpha KO EMSCs (lenti-Cre infected Hif-1af/f). Image collected and cropped by CiteAb from the following publication (//www.nature.com/articles/srep21520) licensed under a CC-BY licence. HIF-2 alpha/EPAS1 Antibody [NB100-122]

Immunohistochemistry: HIF-2 alpha/EPAS1 Antibody Pack [NB100-902HIF2] - Formalin-fixed paraffin-embedded tissue sections of human placenta were probed for HIF-2 alpha/EPAS1 mRNA (ACD RNAScope Probe, catalog #410598; Fast Red chromogen, ACD catalog # 322750). Adjacent tissue section was processed for immunohistochemistry using Rabbit Polyclonal (Novus Biologicals catalog # NB100-122) at 1:100 dilution with one-hour incubation at room temperature followed by incubation with anti-rabbit IgG VisUCyte HRP Polymer Antibody (Catalog # VC003) and DAB chromogen (yellow-brown). Tissue was counterstained with hematoxylin (blue). Specific staining was localized to trophoblastic cells. HIF-2 alpha/EPAS1 Antibody [NB100-122]

Immunohistochemistry: HIF-2 alpha/EPAS1 Antibody Pack [NB100-902HIF2] - Normal ganglia 1 (NG1), normal ganglia 2 (NG2) and IDH-mutant (IDH). Sporadic PGL (Spo. PGL). HIF2 alpha staining. Image collected and cropped by CiteAb from the following publication (//doi.org/10.1371/journal.pone.0127471) licensed under a CC-BY licence. HIF-2 alpha/EPAS1 Antibody (ep190b) [NB100-132]

• Reactivity: Hu, Mu, Rt
• Applications: WB, Simple Western, ELISA, Flow, ICC/IF, IHC, IHC-Fr, IHC-P, IP, ChIP, KO
• Host: Mouse

HIF-2 alpha/EPAS1 Antibody Pack Summary

• Immunogen: For NB100-122: Proprietary peptide derived from the mouse/human HIF-2a. For NB100-132: Human HIF-2a amino acids 535-631.
• Specificity: These antibodies are specific for HIF-2alpha.
• Host: Mouse
• Gene: EPAS1

Applications/Dilutions

• Dilutions:
  • Chromatin Immunoprecipitation (ChIP)
  • ELISA
  • Flow Cytometry
  • Immunocytochemistry/Immunofluorescence
  • Immunohistochemistry
  • Immunohistochemistry-Frozen
  • Immunohistochemistry-Paraffin
  • Immunoprecipitation
  • Knockout Validated
  • Simple Western
  • Western Blot
• Application Notes: By Western blot, these antibodies recognize a band ~118 kDa, representing HIF-2a in induced tissues and cells. The monoclonal has been used for immunohistochemistry. The polyclonal has also been used, but success has varied. Nuclear extracts should be used for Western analysis, if possible.
  See individual datasheets for validated applications.
• Control:

  HIF-2 alpha/EPAS1 Overexpression Lysate

Reactivity Notes

NB 100-122 recognizes human, mouse, and rat HIF-2a. Other species have not been tested.NB 100-132 reacts with human and rat HIF-2 alpha. Results in mouse have been mixed (some + and some -). Other species have not been tested.

Packaging, Storage & Formulations

• Storage: Storage is content dependent.

Notes

This pack contains 1 vial each of: NB100-122SS (0.025 mL) and NB100-132SS (0.05 mL).

Alternate Names for HIF-2 alpha/EPAS1 Antibody Pack

• Basic-helix-loop-helix-PAS protein MOP2
• BHLHE73
• Class E basic helix-loop-helix protein 73
• ECYT4
• endothelial PAS domain protein 1
• endothelial PAS domain-containing protein 1
• EPAS1
• EPAS-1
• HIF 2A
• HIF-1-alpha-like factor
• HIF-1alpha-like factor
• HIF2 alpha
• HIF-2 alpha
• hif2a angiogenesis
• HIF2A
• HIF-2-alpha
• HIF2-alpha
• HLF
• hypoxia-inducible factor 2 alpha
• Hypoxia-inducible factor 2-alpha
• Member of PAS protein 2
• MOP2
• PAS domain-containing protein 2
• PASD2

Background

Hypoxia contributes to the pathophysiology of human disease, including myocardial and cerebral ischemia, cancer, pulmonary hypertension, congenital heart disease and chronic obstructive pulmonary disease (1). In cancer, and particularly solid tumors, hypoxia plays a critical role in the regulation of genes involved in stem cell renewal, epithelial to mesenchymal transition (EMT), metastasis and angiogenesis. In the tumor microenvironment (TME), hypoxia influences the properties and function of stromal cells (e.g., fibroblasts, endothelial and immune cells) and is a strong determinant of tumor progression (2,3).

HIF-1 or hypoxia inducible factor 1, is a transcription factor commonly referred to as a "master regulator of the hypoxic response" for its central role in the regulation of cellular adaptations to hypoxia. Similarly, HIF-2 alpha plays a role in cellular responses to hypoxia, but whereas HIF-1 alpha is ubiquitously expressed, HIF-2 alpha is predominantly expressed in the vascular endothelium at embryonic stages and after birth in select cells and tissue types (e.g., fibroblasts, hepatocytes and myocytes at 96kDa) (4). Following a similar mechanism to HIF-1 alpha, HIF-2 alpha is stabilized under hypoxic conditions by the formation of a heterodimer with an ARNT/HIF-1 beta subunit. Stable HIF-2 alpha-ARNT/HIF-1 beta heterodimers engage p300/CBP in the nucleus for binding to hypoxic response elements (HREs), inducing transcription, and thus regulation of genes (e.g., EPO, VEGFA). HIF-1 predominantly transactivates genes involved in glycolytic control and pro-apoptotic genes (e.g., LDHA and BNIP3), and HIF-2 regulates the expression of genes involved in invasion and stemness (e.g., MMP2, and OCT4). Common gene targets for HIF-1 and HIF-2 include VEGFA and GLUT1 (5).

The HIF-2 alpha subunit is rapidly targeted and degraded by the ubiquitin proteasome system under normoxic conditions. This process is mediated by oxygen-sensing enzymes, prolyl hydroxylase domain enzymes (PHDs), which catalyze the hydroxylation of key proline residues (Pro-405 and Pro-531) within the oxygen-dependent degradation domain of HIF-2 alpha (5). Once hydroxylated, HIF-2 alpha binds the von Hippel-Lindau tumor suppressor protein (pVHL) for subsequent ubiquitination and proteasomal degradation (5,6).

References

1. Semenza, G. L., Agani, F., Feldser, D., Iyer, N., Kotch, L., Laughner, E., & Yu, A. (2000). Hypoxia, HIF-1, and the pathophysiology of common human diseases. Advances in Experimental Medicine and Biology.

2.Muz, B., de la Puente, P., Azab, F., & Azab, A. K. (2015). The role of hypoxia in cancer progression, angiogenesis, metastasis, and resistance to therapy. Hypoxia. https://doi.org/10.2147/hp.s93413

3. Huang, Y., Lin, D., & Taniguchi, C. M. (2017). Hypoxia inducible factor (HIF) in the tumor microenvironment: friend or foe? Science China Life Sciences. https://doi.org/10.1007/s11427-017-9178-y

4. Hu, C.-J., Wang, L.-Y., Chodosh, L. A., Keith, B., & Simon, M. C. (2003). Differential Roles of Hypoxia-Inducible Factor 1 (HIF-1) and HIF-2 in Hypoxic Gene Regulation. Molecular and Cellular Biology. https://doi.org/10.1128/mcb.23.24.9361-9374.2003

5. Koh, M. Y., & Powis, G. (2012). Passing the baton: The HIF switch. Trends in Biochemical Sciences. https://doi.org/10.1016/j.tibs.2012.06.004

6. Koyasu, S., Kobayashi, M., Goto, Y., Hiraoka, M., & Harada, H. (2018). Regulatory mechanisms of hypoxia-inducible factor 1 activity: Two decades of knowledge. Cancer Science. https://doi.org/10.1111/cas.13483

Limitations

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

Publications for HIF-2 alpha/EPAS1 Antibody Pack (NB100-902HIF2)(12)

Publications using NB100-902HIF2

• Hill, P et al. Inhibition of hypoxia inducible factor hydroxylases protects against renal ischemia-reperfusion injury. J Am Soc Nephrol;19(1):39-46. 2008 Jan. [PMID: 18178798]
• Inoue, Y et al. Expression of hypoxia-inducible factor 1alpha and 2alpha in choroidal neovascular membranes associated with age-related macular degeneration. Br J Ophthalmol;91(12):1720-1. 2007 Dec. [PMID: 18024825]
• Khacho, M et al. Cancer-causing mutations in a novel transcription-dependent nuclear export motif of VHL abrogate oxygen-dependent degradation of hypoxia-inducible factor. Mol Cell Biol;28(1):302-14. 2008 Jan. [PMID: 17967880]
• Fukuda R, Zhang H, Kim JW, Shimoda L, Dang CV, Semenza GL. HIF-1 regulates cytochrome oxidase subunits to optimize efficiency of respiration in hypoxic cells. Cell;129(1):111-22. 2007 Apr 6. [PMID: 17418790]
• Shinojima, T et al. Renal cancer cells lacking hypoxia inducible factor (HIF)-1alpha expression maintain vascular endothelial growth factor expression through HIF-2alpha. Carcinogenesis;28(3):529-36. 2007 Mar. [PMID: 16920734]
• Nakamura, K et al. Brain-derived neurotrophic factor activation of TrkB induces vascular endothelial growth factor expression via hypoxia-inducible factor-1alpha in neuroblastoma cells. Cancer Res;66(8):4249-55. 2006 Apr 15. [PMID: 16618748]
• Mizukami, Y et al. Hypoxic regulation of vascular endothelial growth factor through the induction of phosphatidylinositol 3-kinase/Rho/ROCK and c-Myc. J Biol Chem;281(20):13957-63. 2006 May 19. [PMID: 16543245]
• Hui, AS et al. Calcium signaling stimulates translation of HIF-alpha during hypoxia. FASEB J;20(3):466-75. 2006 Mar. [PMID: 16507764]
• Schultz, K, Fanburg, BL, Beasly, D. Hypoxia and hypoxia-inducible factor-1alpha promote growth factor-induced proliferation of human vascular smooth muscle cells. Am J Physiol Heart Circ Physiol;290(6):H2528-34. 2006 Jun. [PMID: 16399861]
• Calvani, M et al. Hypoxic induction of an HIF-1alpha-dependent bFGF autocrine loop drives angiogenesis in human endothelial cells. Blood;107(7):2705-12. 2006 Apr 1. [PMID: 16304044]
• Mascio, CE et al. Myocardial vascular and metabolic adaptations in chronically anemic fetal sheep. Am J Physiol Regul Integr Comp Physiol;289(6):R1736-45. 2005 Dec. [PMID: 16123231]
• Kuznetsova, AV et al. von Hippel-Lindau protein binds hyperphosphorylated large subunit of RNA polymerase II through a proline hydroxylation motif and targets it for ubiquitination. Proc Natl Acad Sci U S A;100(5):2706-11. 2003 Mar 4. [PMID: 12604794]

Review for HIF-2 alpha/EPAS1 Antibody Pack (NB100-902HIF2) (1)

Average Rating: 4

(Based on 1 review)

reviewed by: Blossom Damania WB 05/05/2010

Summary

• Application: Western Blot

FAQs for HIF-2 alpha/EPAS1 Antibody Pack (NB100-902HIF2). (Showing 1 - 2 of 2 FAQs).

1. I am planning to order the hif2a antibody pack NB100-902HIF2. Do you know if the antibodies can be re-used? Is a 1:500 dilution recommended for ecl detection? Will the antibodies work in total cell extracts?
   • We do not recommend re-using the antibodies and we will not guarantee their function if they are re-used. You can use a normal dilution of secondary antibody for use with these antibodies. We have only tested their use on whole cell lysates. Since HIF2a is a nuclear protein, you will want to make sure it will still be in your cell extracts.
2. I would like to know the working concentration for the 2 HIF-2 alpha antibodies in the kit NB100-902HIF2.
   • The working dilutions for Western Blots are: NB100-122 we recommend a range of 1:200-1:1000 depending on your samples, and NB100-132 we recommend 1:500. They both come at a starting concentration of 1.0 mg/mL. If you have questions on other applications let us know. Additionally, all of the dilutions can be found on the respective datasheets should you need a quicker answer for the working concentration.

Bioinformatics

• Gene Symbol: EPAS1