HIF-1 alpha Antibody - BSA Free

Images

 
Biological Strategies: Western Blot: HIF-1 alpha Antibody [NB100-654] - Effects of in vitro hypoxia on the expression of mouse HIF-1 alpha in HIF-1 alpha+/- and HIF-1 alpha+/+ astrocytes. Confluent astrocyte ...read more
Biological Strategies: Western Blot: HIF-1 alpha Antibody [NB100-654] - Effects of CoCl2 treatment on HIF-1 alpha expression in mouse astrocytes. Western blots using nuclear proteins show that both hypoxia (H) ...read more
Immunohistochemistry-Paraffin: HIF-1 alpha Antibody [NB100-654] - IHC analysis of a formalin-fixed paraffin-embedded tissue section of human endometrium carcinoma AN3CA cell line based xenograft using rabbit polyclonal ...read more
Biological Strategies: Western Blot: HIF-1 alpha Antibody [NB100-654] - HIF1-alpha analysis using NB100-654. Image submitted from verified customer review.
Biological Strategies: Western Blot: HIF-1 alpha Antibody [NB100-654] - Detection of HIF-1 alpha in cobalt chloride treated/untreated COS-7 nuclear extracts using NB100-654.
Simple Western: HIF-1 alpha Antibody [NB100-654] - Simple Western lane view shows a specific band for HIF-1 alpha in 0.5 mg/ml of Hypoxic HeLa lysate. This experiment was performed under reducing conditions using the ...read more
Gel Super Shift Assays: HIF-1 alpha Antibody [NB100-654] - Mouse HIF-1 alpha+/+ and HIF-1 alpha+/- cells were exposed to hypoxia or 125 uM CoCl2 for 6 h, nuclear extracts were then prepared and EMSA carried out. HIF-1 ...read more

Product Details

Summary
Reactivity Hu, Mu, Rt, Po, BvSpecies Glossary
Applications WB, Simple Western, GS, ICC/IF, IHC, KD
Clonality
Polyclonal
Host
Rabbit
Conjugate
Unconjugated
Format
BSA Free
Concentration
1.0 mg/ml

Order Details

HIF-1 alpha Antibody - BSA Free Summary

Immunogen
This HIF-1 alpha Antibody was developed against a fusion protein containing amino acids 432 - 528 of human HIF-1 alpha [Uniprot# Q16665].
Localization
Nuclear
Isotype
IgG
Clonality
Polyclonal
Host
Rabbit
Gene
HIF1A
Purity
Immunogen affinity purified
Innovator's Reward
Test in a species/application not listed above to receive a full credit towards a future purchase.

Applications/Dilutions

Dilutions
  • Gel Super Shift Assays
  • Immunocytochemistry/ Immunofluorescence 1:10 - 1:500. Use reported in scientific literature
  • Immunohistochemistry 1:100 - 1:300
  • Immunohistochemistry-Frozen 1:100 - 1:300. Use reported in scientific literature
  • Immunohistochemistry-Paraffin 1:100 - 1:300. Use reported in scientific literature
  • Knockdown Validated reported in scientific literature (PMID 32054096)
  • Simple Western 1:100
  • Western Blot 1:1000
Application Notes
Nuclear extracts are recommended.

In Simple Western only 10 - 15 uL of the recommended dilution is used per data point. Separated by Size-Wes, Sally Sue/Peggy Sue.
Theoretical MW
93 kDa.
Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
Control
COS-7 Nuclear Hypoxic Induced Cell Lysate
Human Placenta Whole Tissue Lysate (Adult Whole Normal)
Human Bone Marrow Whole Tissue Lysate (Adult Whole Diabetes)
HeLa CoCl2 treated/Untreated Cell Lysate
HepG2 CoCl2 treated/Untreated Cell Lysate
HeLa Hypoxic / Normoxic Cell Lysate
HepG2 Hypoxic / Normoxic Cell Lysate
HIF-1 alpha Knockout CoCl2-treated/untreated HeLa Cell Lysate
HIF-1 alpha Knockout Hypoxic-treated/untreated HeLa Cell Lysate
NIH 3T3 CoCl2 treated/Untreated Cell Lysate
L929 CoCl2 treated/Untreated Cell Lysate
Raw 264.7 CoCl2 treated/Untreated Cell Lysate
PC12 CoCl2 treated/Untreated Cell Lysate
PC12 Hypoxic / Normoxic Cell Lysate
Raw 264.7 Hypoxic / Normoxic Cell Lysate
L929 Hypoxic / Normoxic Cell Lysate
NIH 3T3 Hypoxic / Normoxic Cell Lysate
Reviewed Applications
Read 1 Review rated 5
using
NB100-654 in the following applications:

Publications
Read Publications using
NB100-654 in the following applications:

Reactivity Notes

Mouse reactivity was reported in scientific literature (PMID: 23959856). Rat reactivity was reported in scientific literature (PMID: 24122166). Use in Bovine reported in scientific literature (PMID:32054096).

Packaging, Storage & Formulations

Storage
Aliquot and store at -20C or -80C. Avoid freeze-thaw cycles.
Buffer
PBS
Preservative
0.02% Sodium Azide
Concentration
1.0 mg/ml
Purity
Immunogen affinity purified

Alternate Names for HIF-1 alpha Antibody - BSA Free

  • AINT
  • anti-HIF-1 alpha
  • anti-HIF1A
  • ARNT interacting protein
  • ARNT-interacting protein
  • Basic-helix-loop-helix-PAS protein MOP1
  • BHLHE78
  • Class E basic helix-loop-helix protein 78
  • HIF 1A
  • HIF1 alpha
  • HIF-1 alpha
  • HIF1
  • HIF1A
  • HIF-1a
  • HIF-1alpha
  • HIF-1-alpha
  • HIF1-alpha
  • hypoxia inducible factor 1 alpha subunit, hypoxia inducible factor 1 subunit alpha
  • hypoxia inducible factor 1, alpha subunit (basic helix-loop-helix transcription factor)
  • hypoxia-inducible factor 1-alpha
  • Member of PAS protein 1
  • member of PAS superfamily 1
  • MOP1
  • PAS domain-containing protein 8
  • PASD8

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 (predicted molecular weight 93kDa), 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. In its active form under hypoxic conditions, HIF-1 is stabilized by the formation of a heterodimer of HIF-1 alpha and ARNT/HIF-1 beta subunits. Nuclear HIF-1 engages p300/CBP for binding to hypoxic response elements (HREs). This process induces transcription and regulation of genes including EPO, VEGF, iNOS2, ANGPT1 and OCT4 (4,5).

Under normoxic conditions, the HIF-1 alpha subunit is rapidly targeted and degraded by the ubiquitin proteasome system. This process is mediated by prolyl hydroxylase domain enzymes (PHDs), which catalyze the hydroxylation of key proline residues (Pro-402 and Pro-564) within the oxygen-dependent degradation domain of HIF-1 alpha. Once hydroxylated, HIF-1 alpha binds the von Hippel-Lindau tumor suppressor protein (pVHL) for subsequent ubiquitination and proteasomal degradation (4). pVHL dependent regulation of HIF-1 alpha plays a role in normal physiology and disease states. Regulation of HIF-1 alpha by pVHL is critical for the suppressive function of FoxP3+ regulatory Tcells (6). Repression of pVHL expression in chronic lymphocytic leukemia (CLL) B cells leads to HIF-1 alpha stabilization and increased VEGF secretion (7).

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. 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

5. Dengler, V. L., Galbraith, M. D., & Espinosa, J. M. (2014). Transcriptional regulation by hypoxia inducible factors. Critical Reviews in Biochemistry and Molecular Biology. https://doi.org/10.3109/10409238.2013.838205

6. Lee, J. H., Elly, C., Park, Y., & Liu, Y. C. (2015). E3Ubiquitin Ligase VHL Regulates Hypoxia-Inducible Factor-1 alpha to Maintain Regulatory T Cell Stability and Suppressive Capacity. Immunity. https://doi.org/10.1016/j.immuni.2015.05.016

7. Ghosh, A. K., Shanafelt, T. D., Cimmino, A., Taccioli, C., Volinia, S., Liu, C. G., ... Kay, N. E. (2009). Aberrant regulation of pVHL levels by microRNA promotes the HIF/VEGF axis in CLL B cells. Blood. https://doi.org/10.1182/blood-2008-10-185686

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.

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Publications for HIF-1 alpha Antibody (NB100-654)(13)

We have publications tested in 5 confirmed species: Human, Mouse, Rat, Bovine, Porcine.

We have publications tested in 6 applications: ICC/IF, IF/IHC, IHC-Fr, IHC-P, KD, WB.


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(1)
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(2)
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(2)
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(3)
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Showing Publications 1 - 10 of 13. Show All 13 Publications.
Publications using NB100-654 Applications Species
Strassheim D, Karoor V, Nijmeh H et al. c-Jun, Foxo3a, and c-Myc Transcription Factors are Key Regulators of ATP-Mediated Angiogenic Responses in Pulmonary Artery Vasa Vasorum Endothelial Cells Cells 2020-02-11 [PMID: 32054096] (KD, WB, Bovine) KD, WB Bovine
Gericke A, Mann C, Zadeh JK et al. Elevated Intraocular Pressure Causes Abnormal Reactivity of Mouse Retinal Arterioles Oxid Med Cell Longev 2019-12-29 [PMID: 31976030] (IF/IHC, Mouse) IF/IHC Mouse
Zadeh, JK;Ruemmler, R;Hartmann, EK;Ziebart, A;Ludwig, M;Patzak, A;Xia, N;Li, H;Pfeiffer, N;Gericke, A; Responses of retinal arterioles and ciliary arteries in pigs with acute respiratory distress syndrome (ARDS) Exp. Eye Res. 2019-04-22 [PMID: 31022399] (IHC-Fr, Porcine) IHC-Fr Porcine
Serganova I, Cohen IJ, Vemuri k et al. LDH-A regulates the tumor microenvironment via HIF-signaling and modulates the immune response. PLoS ONE. 2018-09-24 [PMID: 30248111] (IHC-P, Mouse) IHC-P Mouse
Bouchard G, Therriault H, Geha S et al. Radiation-induced lung metastasis development is MT1-MMP-dependent in a triple-negative breast cancer mouse model. Br J Cancer 2017-02-14 [PMID: 28103615] (Mouse) Mouse
Knudsen ES, Balaji U, Freinkman E et al. Unique metabolic features of pancreatic cancer stroma: relevance to the tumor compartment, prognosis, and invasive potential. Oncotarget. 2016-11-29 [PMID: 27623078] (IF/IHC, Human) IF/IHC Human
De Bruycker S, Vangestel C, Van den Wyngaert T et al. Baseline [(18)F]FMISO microPET as a Predictive Biomarker for Response to HIF-1alpha Inhibition Combined with 5-FU Chemotherapy in a Human Colorectal Cancer Xenograft Model. Mol Imaging Biol. 2016-01-04 [PMID: 26728163] (IHC-P, Human) IHC-P Human
Flann KL, Rathbone CR, Cole LC et al. Hypoxia simultaneously alters satellite cell-mediated angiogenesis and hepatocyte growth factor expression. J Cell. Physiol. 2014-05-01 [PMID: 24122166] (WB, Rat)

Details:
HIF1 alpha antibody used for WB on cytoplasmic and nuclear extractions of normoxia, hypoxia or uM 150 CoCl2 exposed primary satellite cells which were isolated from male Sprague Dawley rats (Figure 4).
WB Rat
Cao Y, Eble JM, Moon E et al. Tumor Cells Upregulate Normoxic HIF-1alpha in Response to Doxorubicin. Cancer Res. 2013-08-19 [PMID: 23959856] (WB, Mouse, Human) WB Mouse, Human
Chiang C-K, Tanaka T, Inagi R et al. Indoxyl sulfate, a representative uremic toxin, suppresses erythropoietin production in a HIF-dependent manner. Lab Invest. 2011-08-22 [PMID: 21863063]
Show All 13 Publications.

Review for HIF-1 alpha Antibody (NB100-654) (1) 51

Average Rating: 5
(Based on 1 review)
We have 1 review tested in 1 species: Human.

Reviews using NB100-654:
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Images Ratings Applications Species Date Details
Western Blot HIF-1 alpha NB100-654
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5
reviewed by:
Ming Lu
WB Human 07/25/2014
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Summary

ApplicationWestern Blot
Sample Testedhuman cancer lines
SpeciesHuman
LotC-2

Product General Protocols

View specific protocols for HIF-1 alpha Antibody (NB100-654): Find general support by application which include: protocols, troubleshooting, illustrated assays, videos and webinars.

Video Protocols

WB Video Protocol
ICC/IF Video Protocol

FAQs for HIF-1 alpha Antibody (NB100-654). (Showing 1 - 10 of 11 FAQs).

  1. Why is there a difference between the theoretical MW for HIF1A and the observed MW for HIF-1 alpha?
    • HIF1A, like many other proteins, has post-translational modifications. Depending on the size, amount and nature of the post-translational modifications, it can cause subtle to very large changes in molecular weight.
  2. Which antibody(ies) do you recommend for the detection of HIF-1a by immunohistochemistry in the sections of paraffin-embedded mouse liver samples? I would appreciate if you can give me several choices and rank them in the order of performance. My goal is to distinguish HIF upregulation by prolyl hydroxylase inhibitor in different liver cells.
    • All of our antibodies are of high quality and are well tested/validated in species/applications we list on the datasheet. However, we suggest the following four HIF-1 alpha antibodies based upon customer reviews, as well as the number of peer reviewed publications in which these products have been cited by researchers from reputed institutes. (1) HIF-1 alpha Antibody (H1alpha67) (cat# NB100-105) (cited in at least 218 peer reviewed publications) (2) HIF-1 alpha Antibody (cat# NB100-479) (cited in at least 51 peer reviewed publications) (3) HIF-1 alpha Antibody (H1alpha67) (cat# NB100-123 ) (cited in at least 38 peer reviewed publications) (4) HIF-1 alpha Antibody (cat# NB100-449) (cited in at least 31 peer reviewed publications).
  3. I would like to know, does a path exist for detection of HIF 1 in venous blood before and after revascularization of the leg? 
    • We are not entirely sure if HIF-1 alpha will be present in the leg after revascularization. It may be present, but you may want to search the literature to see if this has been looked at before. If not, then this would certainly be an experiment worth doing.
  4. What is the molecular weight (kDa) of protein HIF 1 alpha in western blot?
    • The theoretical molecular weight of HIF 1-alpha is ~93kDa. However, you will likely see a band between 100-120kDa due to phosphorylation.
  5. We got the Hif1a (NB100-105) antibody from you guys. I used the concentration that is mentioned on your website, but I am getting a band of a completely different size (~70kDa) and not the 120 kDa mentioned.
    • HIF-1 alpha is a notoriously difficult protein to work with due to its rapid degradation. Therefore, the ~70kDa bands are most likely degradation products. It is very important to lyse the cells in hypoxic conditions. We strongly recommend lysing the cells directly into the Laemmli buffer and doing that quickly, so that the exposure to oxygen is minimized.Please go through our hypoxia related FAQs, you should find them very informative.Also, running a positive control may help confirm the band specificity in your samples. You may prepare them yourself or choose some from our catalog, for example: 1) HeLa Hypoxic / Normoxic Cell Lysate (NBP2-36452)2) HeLa Hypoxic (CoCl2) / Normoxic Cell Lysate (NBP2-36450)
  6. I performed several Western Blots of HIF-1 alpha with different lysis buffers, whole lysates, and cytoplasm/nuclei extractions. I can’t seem to get a good western blot (poor signal, band much lower than expected, etc.). Can someone suggest some technical considerations/tricks I should consider using?
    • A major issue that researchers working with HIF-1 alpha is degradation due to exposure to oxygen. In western blot, this results in a weaker band and/or the appearance of multiple low molecular weight bands (40-80 kDa). We recommend preparing the lysates after collection of cells/tissues as quickly as possible (on ice), preferably in a hypoxic chamber. We also recommend including a true hypoxia mimetic control (eg: cells treated with CoCl2, DMOG… etc.). The controls help distinguish your band of interest from potential degradation/dimer bands.For more troubleshooting tips and frequently asked questions regarding hypoxia/HIFs, you can refer to our hypoxia-related FAQs.
  7. I am doing HIF1 westerns in HIF-overexpressing mouse liver and adipose tissue using Novus antirabbit HIF1a antibody with overnight incubation. I am getting strong bands around 90kDa. I am aware that HIF theoretical molecular weight is 93kDa, but in westerns, the HIF band is usually around 120kDa according to my internet research. Can someone let me know if I’m getting the right HIF band or just some non-specific bands? Thanks.
    • (1)    HIF-1 alpha’s theoretical molecular weight is 93kDa. The post translationally modified/ubiquitinated form of HIF-1 alpha protein (fails to undergo proteasomal degradation) shows up as a band in the 110-130 kDa range on a Western blot.(2)    The dimeric protein may appear at a position above 200 kDa on non-reducing gels.(3)    Importantly, HIFs are among the most rapidly degradable proteins; therefore, sample preparation is highly important when analyzing HIF1 alpha or HIF2 alpha. When degraded, HIF-1 alpha may show up between 40-80 kDa position on Western blot. Degradation may be avoided by preparing the samples as soon as possible after collection of cells/tissues in hypoxic chamber. Notably, the tissues/cells should be kept on ice during lysate preparation and the lysates should be analyzed as soon as possible.(4)    For troubleshooting suggestions/feedback on more than 25 similar frequently asked questions, I would recommend visiting Novus page: FAQs - Hypoxia and HIFs (5)    Last but not the least, Novus technical support team may be contacted via email
  8. I have Hif1a nuclear protein extract at -80C. I am wondering if anyone knows how long it would be good for at that temperature since HIf1a is known to be degraded easily.Thank you!
    • You could try a few things to further inhibit the degradation.1) Use the protease inhibitors (if you are not already using them).2) Lyse cells into a buffer that contains SDS or LDS (eg: Laemmli's buffer), since SDS and LDS denature and inhibit proteases. Lysis may even be performed with reducing agents in the buffer (eg. DTT), but this will make your lysates unsuitable for BCA assay.3) Lysing samples rapidly ensures that the samples are instantly homogenized (it also shears DNA released by the SDS).5) Flash-freezing samples in liquid nitrogen rather than freezing at -80*C reduces the window of time for protease activity.6) Freeze samples in individual aliquots, instead of thawing the same vial multiple times.
  9. I am curious to know the biochemical reactions of CoCl2 that mimic hypoxia. Is it that CoCl2 can bind any ubiquitin enzyme which regulates their degradation?
    • CoCl2 inhibits PHD enzymes (the body’s “oxygen sensors”) by replacing the Fe ion with Co, preventing these enzymes from marking HIF-1 alpha for degradation. CoCl2-based hypoxia mimetic samples are often used as positive control in HIF analysis. For more troubleshooting tips and frequently asked questions regarding hypoxia/HIFs, you can refer to our hypoxia-related FAQs.
  10. I am curious to know the biochemical reactions of CoCl2 that mimic hypoxia. Is it that CoCl2 can bind any ubiquitin enzyme which regulates their degradation?
    • CoCl2 inhibits PHD enzymes (the body’s “oxygen sensors”) by replacing the Fe ion with Co, preventing these enzymes from marking HIF-1 alpha for degradation. CoCl2-based hypoxia mimetic samples are often used as positive control in HIF analysis. For more troubleshooting tips and frequently asked questions regarding hypoxia/HIFs, you can refer to our hypoxia-related FAQs.
  11. Show All 11 FAQs.

Control Lysate(s)

Secondary Antibodies

 

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Blogs on HIF-1 alpha. Showing 1-10 of 37 blog posts - Show all blog posts.

Hypoxia-Dependent CAR Stabilizing Construct in T cells Improves Solid Tumor Targeting and Efficacy
By Victoria Osinski, PhDDespite advances in the development of cancer immunotherapies, those specifically targeting tumors still remains limited. Currently, there is great interest in utilizing chimeric antigen rece...  Read full blog post.

Tired T cells: Hypoxia Drives T cell Exhaustion in the Tumor Microenvironment
By Hunter MartinezThe paradigm shifting view of the immune system being leveraged to target cancer has led to numerous therapeutic breakthroughs. One major cell group responsible for this revelation is a T cell. ...  Read full blog post.

Understanding ‘Y’ in Breast Cancer: Crucial Role of DNA/RNA-binding Protein YB-1 in the Development, Pre-Invasive, and Metastatic Phases
Jamshed Arslan, Pharm D, PhD In the United States, 1 in 8 women will be diagnosed with breast cancer in her lifetime.1 Despite the prevalence, cancer genesis is a mystery. The heterogeneity of cancers makes it diff...  Read full blog post.


  Read full blog post.

Breast cancer stem cells survive chemotherapy through S100A10-ANXA2-SPT6 interaction that epigenetically promotes OCT4-mediated stemness
By Jamshed Arslan, Pharm D, PhDBreast cancer is the most common cancer among women that causes the greatest number of cancer-related deaths worldwide. After radiotherapy or cytotoxic chemotherapy like paclitax...  Read full blog post.

mTOR Signaling and the Tumor Microenvironment
By Yoskaly Lazo-Fernandez, PhD The mammalian target of rapamycin (mTOR) is a conserved serine/threonine kinase that, as a member of two distinct intracellular protein complexes, mTORC1 and mTORC2, regulates protein ...  Read full blog post.

Bad news for stomach cancer: BAMBI protein inhibits gastric carcinoma via TGF-beta/epithelial-mesenchymal transition signaling
By Jamshed Arslan Pharm.D. Gastric carcinoma is the second leading cause of cancer-related deaths worldwide. One of the key features of gastric carcinoma is acidosis, which promotes growth and metastasis of gastric ...  Read full blog post.

Developmental regulator Daam2 promotes glial cell tumors by degrading Von Hippel-Lindau protein
By Jamshed Arslan Pharm.D. Glioblastoma is an aggressive type of cancer that forms from the star-shaped glial cells of the central nervous system, called astrocytes. Intriguingly, several genes linked to glioblasto...  Read full blog post.

Stemness for Surviving Hypoxia: TGF-beta/Smad Signaling in Multiple Myeloma
By Jamshed Arslan Pharm.D. Multiple myeloma (MM) is a cancer of antibody-producing plasma cells. The bone marrow (BM) of MM patients is hypoxic, and MM cells overexpress many cancerous genes that are regulated by hy...  Read full blog post.

Forecasting and Targeting a Rare Cancer with Hypoxia-Inducible Factor
By Jamshed Arslan Pharm.D. Cancers of nerve, adipose, and other soft tissues are called soft tissue sarcomas (STS). Malignant peripheral nerve sheath tumor (MPNST) is an example of a rare and hard-to-treat STS; eve...  Read full blog post.

Showing 1-10 of 37 blog posts - Show all blog posts.
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Ming Lu
07/25/2014
Application: WB
Species: Human

Bioinformatics

Gene Symbol HIF1A