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15792
Hypoxia Pathway Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit

Hypoxia Pathway Antibody Sampler Kit #15792

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other Image 1 - Hypoxia Pathway Antibody Sampler Kit

Chromatin immunoprecipitations were performed with cross-linked chromatin from MCF7 cells treated with cobalt chloride (100 μM) overnight and HIF-1α (D1S7W) XP® Rabbit mAb, using SimpleChIP® Plus 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 ARRDC3, a known target gene of HIF-1α (see additional figure containing ChIP-qPCR data).

other Image 2 - Hypoxia Pathway Antibody Sampler Kit

Confocal immunofluorescent analysis of HeLa cells, treated with either 10 μM MG132 (left) or 10 μM MG132 and 1 mM DMOG (right), using Hydroxy-HIF-1α (Pro564) (D43B5) XP® Rabbit mAb (green). Actin filaments have been labeled using DY-554 phalloidin (red).

other Image 3 - Hypoxia Pathway Antibody Sampler Kit

Chromatin immunoprecipitations were performed with cross-linked chromatin from Hep3B2.1-7 cells treated with cobalt chloride (100 μM) overnight and either HIF-2α (D6T8V) Rabbit mAb #59973 or Normal Rabbit IgG #2729 using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. The enriched DNA was quantified by real-time PCR using SimpleChIP® Human PAI-1 Promoter Primers #33070, human EPO 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.

other Image 4 - Hypoxia Pathway Antibody Sampler Kit

Western blot analysis of extracts from various cell types using FIH (D19B3) Rabbit mAb.

other Image 5 - Hypoxia Pathway Antibody Sampler Kit

Western blot analysis of extracts from various cell types using PHD-2/Egln1 (D31F11) Rabbit mAb.

other Image 6 - Hypoxia Pathway Antibody Sampler Kit

Western blot analysis of extracts from various cell lines using VHL Antibody (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower). Note that 786-O is a VHL-null cell line, demonstrating specificity of the antibody.

other Image 7 - Hypoxia Pathway Antibody Sampler Kit

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.

other Image 8 - Hypoxia Pathway Antibody Sampler Kit

Chromatin immunoprecipitations were performed with cross-linked chromatin from MCF7 cells treated with cobalt chloride (100 μM) overnight and HIF-1α (D1S7W) XP® Rabbit mAb, using SimpleChIP® Plus 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 chromosome 5 (upper), including ARRDC3 (lower), a known target gene of HIF-1α (see additional figure containing ChIP-qPCR data).

other Image 9 - Hypoxia Pathway Antibody Sampler Kit

Western blot analysis of extracts from HeLa cells, treated with either 10 μM of MG132 (to accumulate hydroxylated HIF-1α) or 10 µM MG132 and 1 mM DMOG (to accumulate nonhyroxylated HIF-1α), using Hydroxy-HIF-1α (Pro564) (D43B5) XP® Rabbit mAb (upper) or total HIF-1α Antibody #3716 (lower).

other Image 10 - Hypoxia Pathway Antibody Sampler Kit

Immunohistochemical analysis of paraffin-embedded human breast carcinoma using HIF-1β/ARNT (D28F3) XP® Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).

other Image 11 - Hypoxia Pathway Antibody Sampler Kit

Chromatin immunoprecipitations were performed with cross-linked chromatin from T47D cells treated with BNF (1μM) for 45 min and either HIF-1β/ARNT (D28F3) 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 NFE2L2 Intron 1 Primers #81126, human CYP1A1 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.

other Image 12 - Hypoxia Pathway Antibody Sampler Kit

Western blot analysis of extracts from 786-O and A498 cells using HIF-2α (D6T8V) Rabbit mAb.

other Image 13 - Hypoxia Pathway Antibody Sampler Kit

Chromatin immunoprecipitations were performed with cross-linked chromatin from MCF7 cells treated with cobalt chloride (100 μM, overnight) and either HIF-1α (D1S7W) XP® Rabbit mAb or Normal Rabbit IgG #2729, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. The enriched DNA was quantified by real-time PCR using human ARRDC3 downstream primers, SimpleChIP® Human ERRFI1 Upstream Primers #31180, 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.

other Image 14 - Hypoxia Pathway Antibody Sampler Kit

Immunohistochemical analysis of paraffin-embedded human lung carcinoma using HIF-1β/ARNT (D28F3) XP® Rabbit mAb.

other Image 15 - Hypoxia Pathway Antibody Sampler Kit

Western blot analysis of Hep G2 cells, untreated (-) or treated with cobalt chloride (CoCl2, 100 μM, 4 hr, +) using HIF-2α (D6T8V) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

other Image 16 - Hypoxia Pathway Antibody Sampler Kit

Flow cytometric analysis of U-2 OS cells, untreated (blue) or treated with DMOG (1 mM, 6 h; green), using HIF-1α (D1S7W) XP® Rabbit mAb (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.

other Image 17 - Hypoxia Pathway Antibody Sampler Kit

Immunohistochemical analysis of paraffin-embedded mouse colon using HIF-1β/ARNT (D28F3) XP® Rabbit mAb.

other Image 18 - Hypoxia Pathway Antibody Sampler Kit

Confocal immunofluorescent analysis of Hep G2 cells, untreated (left) or treated with cobalt chloride (500 μM, 24 h; right), using HIF-1α (D1S7W) XP® Rabbit mAb (green). Actin filaments were labeled with DyLight™ 554 Phalloidin #13054 (red).

other Image 19 - Hypoxia Pathway Antibody Sampler Kit

Western blot analysis of extracts from various cell types using HIF-1β/ARNT (D28F3) XP® Rabbit mAb.

other Image 20 - Hypoxia Pathway Antibody Sampler Kit

Immunoprecipitation of HIF-1α from lysate of Hep G2 cells treated with cobalt chloride (100 µM, 4 h). Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is HIF-1α (D1S7W) XP® Rabbit mAb. Western blot analysis was performed using HIF-1α (D1S7W) XP® Rabbit mAb. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as the secondary antibody.

other Image 21 - Hypoxia Pathway Antibody Sampler Kit

Western blot analysis of extracts from Hep G2 cells untreated (-) or treated with cobalt chloride (100 µM, 4 h; +), Raji cells untreated (-) or treated with cobalt chloride (100 µM, 4 h; +) and U-2 OS cells untreated (-) or treated with DMOG (1 mM, 6 h; +) using HIF-1α (D1S7W) XP® Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

To Purchase # 15792T
Product # Size Price
15792T
1 Kit  (7 x 20 µl) $ 500

Product Includes Quantity Applications Reactivity MW(kDa) Isotype
HIF-1α (D1S7W) XP® Rabbit mAb 36169 20 µl
  • WB
  • IP
  • IF
  • F
  • ChIP
H M Mk 120 Rabbit IgG
Hydroxy-HIF-1α (Pro564) (D43B5) XP® Rabbit mAb 3434 20 µl
  • WB
  • IP
  • IF
H Mk 120 Rabbit IgG
HIF-1β/ARNT (D28F3) XP® Rabbit mAb 5537 20 µl
  • WB
  • IP
  • IHC
  • ChIP
H M R Mk 87 Rabbit IgG
HIF-2α (D6T8V) Rabbit mAb 59973 20 µl
  • WB
  • ChIP
H 120 Rabbit IgG
FIH (D19B3) Rabbit mAb 4426 20 µl
  • WB
H M R Mk 42 Rabbit IgG
PHD-2/Egln1 (D31E11) Rabbit mAb 4835 20 µl
  • WB
  • IP
H M R Mk 50 Rabbit IgG
VHL Antibody 68547 20 µl
  • WB
H 18-22 Rabbit 
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

Product Description

The Hypoxia Pathway Antibody Sampler Kit provides an economical means to investigate select proteins involved in the hypoxia pathway. The kit contains enough primary antibodies to perform two western blot experiments with each primary antibody.

Specificity / Sensitivity

HIF-1α (D1S7W) XP® Rabbit mAb recognizes endogenous levels of total HIF-1α protein. This antibody does not cross-react with HIF-2α protein. Hydroxy-HIF-1α (Pro564) (D43B5) XP® Rabbit mAb detects endogenous levels of HIF-1α only when hydroxylated at Pro564. This antibody may cross-react with other overexpressed proline hydroxylated proteins. HIF-1β/ARNT (D28F3) XP® Rabbit mAb detects endogenous levels of total HIF-1β/ARNT protein. HIF-2α (D6T8V) Rabbit mAb recognizes endogenous levels of total HIF-2α protein. This antibody does not cross-react with HIF-1α protein. FIH (D19B3) Rabbit mAb detects endogenous levels of total FIH protein. PHD-2/Egln1 (D31E11) Rabbit mAb detects endogenous levels of total PHD-2/Egln1 protein. VHL Antibody recognizes endogenous levels of total VHL protein.

Source / Purification

Monoclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Leu478 of human HIF-1α protein, Gly688 of human HIF-2α protein, Tyr35 of human FIH protein, and Val226 of human PHD-2/Egln1 protein. Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to the sequence around Ile479 of human HIF-1β/ARNT protein. Monoclonal antibody is produced by immunizing animals with a synthetic hydroxypeptide corresponding to residues surrounding Pro564 of human HIF-1α. Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human VHL protein.

Background

Hypoxia-inducible factor 1 (HIF1) is a heterodimeric transcription factor that plays a critical role in the cellular response to hypoxia (1). The HIF1 complex consists of two subunits, HIF-1α and HIF-1β, which are basic helix-loop-helix proteins of the PAS (Per, ARNT, Sim) family (2). HIF1 regulates the transcription of a broad range of genes that facilitate responses to the hypoxic environment, including genes regulating angiogenesis, erythropoiesis, cell cycle, metabolism, and apoptosis. The widely expressed HIF-1α is typically degraded rapidly in normoxic cells by the ubiquitin/proteasomal pathway. Under normoxic conditions, HIF-1α is proline hydroxylated leading to a conformational change that promotes binding to the von Hippel-Lindau protein (VHL) E3 ligase complex; ubiquitination and proteasomal degradation follows (3,4). Both hypoxic conditions and chemical hydroxylase inhibitors (such as desferrioxamine and cobalt) inhibit HIF-1α degradation and lead to its stabilization. In addition, HIF-1α can be induced in an oxygen-independent manner by various cytokines through the PI3K-AKT-mTOR pathway (5-7).

HIF-1β is also known as AhR nuclear translocator (ARNT) due to its ability to partner with the aryl hydrocarbon receptor (AhR) to form a heterodimeric transcription factor complex (8). Together with AhR, HIF-1β plays an important role in xenobiotics metabolism (8). In addition, a chromosomal translocation leading to a TEL-ARNT fusion protein is associated with acute myeloblastic leukemia (9). Studies also found that ARNT/HIF-1β expression levels decrease significantly in pancreatic islets from patients with type 2 diabetes, suggesting that HIF-1β plays an important role in pancreatic β-cell function (10).

Hypoxia-inducible factor (HIF) is essential for the cellular response to hypoxia (11,12). There are several isoforms of the HIF α subunit. Studies have found that HIF-1α and HIF-2α expression is increased in some human cancers. HIF-1α has both pro- and anti-proliferative activities, whereas HIF-2α does not possess anti-proliferative activity (12). Therefore, HIF-2α likely plays an important role in tumorigenesis (12,13).

FIH (Factor inhibiting HIF-1, HIF asparagine hydroxylase) is a dioxygen-dependent asparaginyl hydroxylase that modifies target protein function by hydroxylating target protein asparagine residues (14-16). HIF, a transcriptional activator involved in control of cell cycle in response to hypoxic conditions, is an important target for FIH regulation. FIH functions as an oxygen sensor that regulates HIF function by hydroxylating at Asn803 in the carboxy-terminal transactivation domain (CAD) of HIF (17,18). During normoxia, FIH uses cellular oxygen to hydroxylate HIF-1 and prevent interaction of HIF-1 with transcriptional coactivators, including the CBP/p300-interacting transactivator. Under hypoxic conditions, FIH remains inactive and does not inhibit HIF, allowing the activator to regulate transcription of genes in response to low oxygen conditions (17-19). FIH activity is regulated through interaction with proteins, including Siah-1, which targets FIH for proteasomal degradation (20). The Cut-like homeodomain protein CDP can bind the FIH promoter region to regulate FIH expression at the transcriptional level (21). Phosphorylation of HIF at Thr796 also can prevent FIH hydroxylation on Asn803 (22). Potential FIH substrates also include proteins with ankyrin repeat domains, such as Iκ-B, Notch, and ASB4 (23-25).

PHD1 (Egln2), PHD-2 (Egln1), and PHD3 (Egln3) are members of the Egln family of proline hydroxylases. They function as oxygen sensors that catalyze the hydroxylation of HIF on prolines 564 and 402, initiating the first step of HIF degradation through the VHL/ubiquitin pathway (26,27).

  1. Sharp, F.R. and Bernaudin, M. (2004) Nat Rev Neurosci 5, 437-48.
  2. Wang, G.L. et al. (1995) Proc Natl Acad Sci U S A 92, 5510-4.
  3. Jaakkola, P. et al. (2001) Science 292, 468-72.
  4. Maxwell, P.H. et al. (1999) Nature 399, 271-5.
  5. Fukuda, R. et al. (2002) J Biol Chem 277, 38205-11.
  6. Jiang, B.H. et al. (2001) Cell Growth Differ 12, 363-9.
  7. Laughner, E. et al. (2001) Mol Cell Biol 21, 3995-4004.
  8. Walisser, J.A. et al. (2004) Proc Natl Acad Sci U S A 101, 16677-82.
  9. Salomon-Nguyen, F. et al. (2000) Proc Natl Acad Sci U S A 97, 6757-62.
  10. Gunton, J.E. et al. (2005) Cell 122, 337-49.
  11. Kaelin, W.G. (2005) Biochem Biophys Res Commun 338, 627-38.
  12. Toschi, A. et al. (2008) J Biol Chem 283, 34495-9.
  13. Gordan, J.D. and Simon, M.C. (2007) Curr Opin Genet Dev 17, 71-7.
  14. Koivunen, P. et al. (2004) J Biol Chem 279, 9899-904.
  15. Linke, S. et al. (2004) J Biol Chem 279, 14391-7.
  16. Lisy, K. and Peet, D.J. (2008) Cell Death Differ 15, 642-9.
  17. Mahon, P.C. et al. (2001) Genes Dev 15, 2675-86.
  18. Lando, D. et al. (2002) Genes Dev 16, 1466-71.
  19. Lando, D. et al. (2002) Science 295, 858-61.
  20. Fukuba, H. et al. (2007) Biochem Biophys Res Commun 353, 324-9.
  21. Li, J. et al. (2007) Mol Cell Biol 27, 7345-53.
  22. Lancaster, D.E. et al. (2004) Biochem J 383, 429-37.
  23. Ferguson, J.E. et al. (2007) Mol Cell Biol 27, 6407-19.
  24. Cockman, M.E. et al. (2006) Proc Natl Acad Sci U S A 103, 14767-72.
  25. Cockman, M.E. et al. (2009) Mol Cell Proteomics 8, 535-46.
  26. Freeman, R.S. et al. (2003) Mol Cells 16, 1-12.
  27. Villar, D. et al. (2007) Biochem J 408, 231-40.

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