Hypoxia Pathway Antibody Sampler Kit

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Hypoxia Pathway Antibody Sampler Kit

品牌： CST

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产品信息

荧光素标记

Unconjugated

抗原名称

Hypoxia Pathway

简单描述

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.

研究领域

癌症,细胞生物学,表观遗传学,纤维化,代谢,神经科学

背景

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.

研究领域

癌症,细胞生物学,表观遗传学,纤维化,代谢,神经科学

翻译后修饰

hydroxylate

数据库链接

Entrez-Gene ID

3091,7428,405,55662,2034,54583

UniProt ID

Q16665,P40337,P27540,Q9NWT6,Q99814,Q9GZT9

参考图片

Simple Western™ analysis of lysates (0.1 mg/mL) from U-937 cells using VHL Antibody #68547. The virtual lane view (left) shows the target band (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 2-40 kDa separation module.

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).

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).

Western blot analysis of extracts from Hep G2 cells, untreated (-) or treated with cobalt chloride (100 μM, 24 hr; +), using HIF-1α (D1S7W) XP® Rabbit mAb #36169 (green), and β-Actin (8H10D10) Mouse mAb #3700 (red). Anti-rabbit IgG (H+L) (DyLight 800 4X PEG Conjugate) #5151 (green) and Anti-mouse IgG (H+L) (DyLight 680 Conjugate) #5470 (red) were used as secondary antibodies.

Simple Western™ analysis of lysates (0.1 mg/mL) from HepG2 cells treated with cobalt chloride (100 µM, 24 hr) using HIF-1α (D1S7W) XP® Rabbit mAb #36169. The virtual lane view (left) shows the target band (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.

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

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

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

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.

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

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.

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.

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).

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.

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).

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).

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).

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

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.

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.

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.

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

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 DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across ARRDC3, a known target gene of HIF-1α (see additional figure containing ChIP-qPCR data).

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 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 DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #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).

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.

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.

CUT&RUN was performed with MCF7 cells treated with cobalt chloride (100 μM) overnight and HIF-1α (D1S7W) XP® Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA library was prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across STC2, a known target gene of HIF-1α (see additional figure containing CUT&RUN-qPCR data).

CUT&RUN was performed with MCF7 cells treated with cobalt chloride (100 μM) overnight and HIF-1α (D1S7W) XP® Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figures show binding across chromosome 5 (upper), including STC2 (lower), a known target gene of HIF-1α (see additional figure containing CUT&RUN-qPCR data).

CUT&RUN was performed with MCF7 cells treated with cobalt chloride (100 μM) overnight and HIF-1α (D1S7W) 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 STC2 exon 1 primers, human FAM162A 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.

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15792T

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