Revision 7
Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Applications:

WB, IP, IHC-P, ChIP, ChIP-seq, C&R

REACTIVITY:

H M R Mk

SENSITIVITY:

Endogenous

MW (kDa):

87

Source/Isotype:

Rabbit IgG

UniProt ID:

#P27540

Entrez-Gene Id:

405

Product Information

Product Usage Information

For optimal ChIP and ChIP-seq results, use 10 μl of antibody and 10 μg of chromatin (approximately 4 x 106 cells) per IP. This antibody has been validated using SimpleChIP® Enzymatic Chromatin IP Kits.

The CUT&RUN dilution was determined using CUT&RUN Assay Kit #86652.
Application Dilution
Western Blotting 1:1000
Immunoprecipitation 1:50
Immunohistochemistry (Paraffin) 1:50 - 1:200
Chromatin IP 1:50
Chromatin IP-seq 1:50
CUT&RUN 1:50

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Specificity / Sensitivity

HIF-1β/ARNT (D28F3) XP® Rabbit mAb detects endogenous levels of total HIF-1β/ARNT protein.

Species Reactivity:

Human, Mouse, Rat, Monkey

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to the sequence around Ile479 of human HIF-1β/ARNT 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).

  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.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Western Blot Buffer

IMPORTANT: For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

Applications Key

WB: Western Blotting IP: Immunoprecipitation IHC-P: Immunohistochemistry (Paraffin) ChIP: Chromatin IP ChIP-seq: Chromatin IP-seq C&R: CUT&RUN

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

使用に関する制限

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Revision 7
#5537

HIF-1β/ARNT (D28F3) XP® Rabbit mAb

Western Blotting Image 1: HIF-1β/ARNT (D28F3) XP® Rabbit mAb Expand Image
Western blot analysis of extracts from various cell types using HIF-1β/ARNT (D28F3) XP® Rabbit mAb.
No image available
Immunohistochemistry Image 1: HIF-1β/ARNT (D28F3) XP® Rabbit mAb Expand Image
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).
Immunohistochemistry Image 2: HIF-1β/ARNT (D28F3) XP® Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded human lung carcinoma using HIF-1β/ARNT (D28F3) XP® Rabbit mAb.
Immunohistochemistry Image 3: HIF-1β/ARNT (D28F3) XP® Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded mouse colon using HIF-1β/ARNT (D28F3) XP® Rabbit mAb.
Chromatin Immunoprecipitation Image 1: HIF-1β/ARNT (D28F3) XP® Rabbit mAb Expand Image
Chromatin immunoprecipitations were performed with cross-linked chromatin from T47D cells treated with BNF (1μM) for 45 min and HIF-1β/ARNT (D28F3) XP® Rabbit mAb, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads)#9005. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across CYP1A2, a known target gene of HIF-1β (see additional figure containing ChIP-qPCR data). For additional ChIP-seq tracks, please download the product data sheet.
Chromatin Immunoprecipitation Image 2: HIF-1β/ARNT (D28F3) XP® Rabbit mAb Expand Image
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.
CUT and RUN Image 1: HIF-1β/ARNT (D28F3) XP® Rabbit mAb Expand Image
CUT&RUN was performed with T47D cells treated with BNF (1μM) for 45 min and HIF-1β/ARNT (D28F3) XP® Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA library was prepared using DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795. The figure shows binding across PEX11B gene.
CUT and RUN Image 2: HIF-1β/ARNT (D28F3) XP® Rabbit mAb Expand Image
CUT&RUN was performed with T47D cells treated with BNF (1μM) for 45 min and HIF-1β/ARNT (D28F3) XP® Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA library was prepared using DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795. The figures show binding across chromosome 1 (upper), including PEX11B (lower).
CUT and RUN Image 3: HIF-1β/ARNT (D28F3) XP® Rabbit mAb Expand Image
CUT&RUN was performed with T47D cells treated with BNF (1μM) for 45 min and either HIF-1β/ARNT (D28F3) XP® Rabbit mAb or Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN) #66362, using CUT&RUN Assay Kit #86652. The enriched DNA was quantified by real-time PCR using human ERCC6 promoter primers and human DPPA2 intron 1 primers. 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.