Hif 1α

Manufactured by Cayman Chemical

Sourced in United States

HIF-1α is a protein that plays a central role in the cellular response to hypoxia. It functions as a transcription factor that regulates the expression of genes involved in various cellular processes, including angiogenesis, erythropoiesis, and glucose metabolism.

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Lab products found in correlation

Hif 2α, by Novus Biologicals (5 mentions) β actin, by Merck Group (5 mentions) Phospho akt, by Cell Signaling Technology (2 mentions) C myc, by Abcam (2 mentions) Dako real detection system, by Agilent Technologies (2 mentions) Biotinylated donkey anti rat and donkey anti rabbit secondary antibodies, by Dianova (2 mentions) Lc3 nb100 2220, by Novus Biologicals (2 mentions) β actin ac 15, by Merck Group (2 mentions) P110δ epr386, by Abcam (2 mentions) Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (2 mentions) Chemidoc imager, by Bio-Rad (2 mentions) Ripa buffer, by Merck Group (2 mentions) P stat3 d3a7, by Cell Signaling Technology (2 mentions) Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (2 mentions) Total ampk, by Cell Signaling Technology (2 mentions) β actin, by Abcam (2 mentions) Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions) P s6ser240 244, by Cell Signaling Technology (1 mentions) P s6k thr389, by Cell Signaling Technology (1 mentions) β actin, by Cell Signaling Technology (1 mentions)

Close spelling variants of this product

HIF-1α Combo Transcription Factor Assay Kit (2 citations) Anti-HIF-1α (24 citations) HIF-1α antibody (4 citations) HIF-1α (C-Term) Polyclonal Antibody (2 citations) HIF-1α Transcription Factor Assay Kit (11 citations) Anti-HIF-1α antibody (3 citations)

28 protocols using hif 1α

Western Blot Analysis of Protein Signaling

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Protein lysates were prepared in 1% CHAPS lysis buffer with protease inhibitor and phosphatase inhibitor, then quantified by BCA assay (Pierce). Samples were run on 10% gels by SDS-polyacrylamide gel electrophoresis, and proteins were transferred by electrophoretic wet transfer to polyvinylidene fluoride membranes. Membranes were blot for p-TBK Ser172 (D52C2), p-AKT Ser473 (D9E) and Thr308 (D25E6), total AKT (C67E7), p-S6K Thr389, p-S6 Ser240/244, β-actin (all Cell Signaling), and HIF-1α (Cayman Chemicals), all diluted to 1:1000 except for HIF-1α (1:500). Incubation with primary antibody overnight was followed by incubation with horseradish peroxidase-linked antibody to rabbit IgG for 45 min. Enhanced chemiluminescence (Perkin Elmer or Amersham) was used to develop the blots. Scans of the original blots are in Supplementary Fig. 7.

Guak H., Al Habyan S., Ma E.H., Aldossary H., Al-Masri M., Won S.Y., Ying T., Fixman E.D., Jones R.G., McCaffrey L.M, & Krawczyk C.M. (2018). Glycolytic metabolism is essential for CCR7 oligomerization and dendritic cell migration. Nature Communications, 9, 2463.

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Western Blot Analysis of Protein Signaling

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Protein lysates were prepared in RIPA lysis buffer, and separated on 10% SDS–polyacrylamide gel electrophoresis gels by electrophoresis. Subsequently, the lysates were transferred to nitrocellulose membranes and probed with the following antibodies and concentrations: HIF-2α 1:1,000 (Novus #NB100-122), HIF-1α 1:1,000 (Cayman Chemicals #1006421), caspase-3 1:1,000 (Cell Signaling #9662 Danvers, MA, USA), GAPDH 1:2,000 (Cell Signaling #2118), β-tubulin 1:1,500 (Cell Signaling #2146), phospho-4E-BP1 1:1,000 (S65, Cell Signaling #9451), 4E-BP1 1:1,000 (Cell Signaling #9452), phospho-S6K1 1:1,000 (T389, Cell Signaling #9205), c-Myc 1:5,000 (Abcam #32072), S6K1 1:1,000 (Cell Signaling #2708), phospho-AKT 1:1,000 (S473, Cell Signaling #9271), AKT 1:1,000 (Cell Signaling #9272), phospho-EGFR 1:1,000 (Y1068, Cell Signaling #3777), EGFR 1:1,000 (Cell Signaling #4267) ANO1 1:500 (Abcam ab64085), phospho-CAMKIIα 1:1,000 (T286, Cell Signaling #12716) and CAMKII 1:1,000 (Cell Signaling #11945). Uncropped immunoblot images are included in Supplementary Fig. 8.

Nakazawa M.S., Eisinger-Mathason T.S., Sadri N., Ochocki J.D., Gade T.P., Amin R.K, & Simon M.C. (2016). Epigenetic re-expression of HIF-2α suppresses soft tissue sarcoma growth. Nature Communications, 7, 10539.

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Quantifying Protein Levels via Western Blotting

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To quantify relative protein levels, 50 µg of total protein per sample was denatured with 2% β-mercaptoethanol and separated on a 4% to 20% TGX Precast Gel (Catalog No. 456-1095; Bio-Rad) with sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred at 25 V onto an Immobilon-FL polyvinylidene difluoride membrane with a Trans-Blot Turbo (Bio-Rad) for 30 minutes. Membranes were blocked in 5% nonfat milk in TBS and probed with rabbit monoclonal anti–P-gp (1:1000; Catalog No. ab170904) or rabbit polyclonal anti–P-gp antibody (Catalog No. sc8313; Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C, washed, and then incubated in goat anti-rabbit IRDye 800 CW (1:40,000; Li-Cor, Lincoln, NE, USA) for an hour at room temperature. Equal protein loading was confirmed using mouse anti–β-actin (1:10,000; Catalog No. 1978; Sigma-Aldrich) and donkey anti-mouse 680 LT IRDye (1:40,000; Li-Cor). Confirmation of hypoxic conditions in our ischemia-like treatment of the RBE4 cells was determined by measuring HIF-1α (1:500; Catlog No. 10006421; Cayman Chemical Company, Ann Arbor, MI, USA). Blots were imaged and densitometrically analyzed with a Li-Cor Odyssey infrared scanner and Image Studio 2.0 software (Li-Cor).

DeMars K.M., Yang C., Hawkins K.E., McCrea A.O., Siwarski D.M, & Candelario-Jalil E. (2017). Spatiotemporal Changes in P-glycoprotein Levels in Brain and Peripheral Tissues Following Ischemic Stroke in Rats. Journal of Experimental Neuroscience, 11, 1179069517701741.

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Immunohistochemical Analysis of Colon Tissues

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Immunohistochemical analysis was conducted on formalin‐fixed paraffin‐embedded tissues. Colon sections were stained with antibodies specific for β‐catenin (Cell Signaling Technology), BrdU (AbD Serotec), cleaved caspase‐3 (Cell Signaling Technology), CD31 (Dianova), c‐MYC (Santa Cruz Biotechnology), cyclin D1 (Santa Cruz Biotechnology), F4/80 (Life Technologies), iNOS (Abcam), and Ki‐67 (Dako Deutschland GmbH). The antibody‐antigen complexes were detected using biotinylated donkey anti‐rat and donkey anti‐rabbit secondary antibodies (Dianova) and the Dako REAL Detection System (Dako). Immunohistochemical detection of HIF‐1α (Cayman Chemical) was performed as previously described.23 Nuclei were counterstained with hematoxylin. Negative controls were performed by omitting the primary antibody. The average number of positively stained cells within at least six high power fields (HPF, 0.237 mm²) was determined by a blinded independent investigator.

Rohwer N., Kühl A.A., Ostermann A.I., Hartung N.M., Schebb N.H., Zopf D., McDonald F.M, & Weylandt K. (2020). Effects of chronic low‐dose aspirin treatment on tumor prevention in three mouse models of intestinal tumorigenesis. Cancer Medicine, 9(7), 2535-2550.

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Western Blot Analysis of DNA Damage Response

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Cells were either lysed in RIPA buffer or nuclear extraction buffer. Proteins were size-fractionated by SDS-PAGE and wet-transfer blotted onto a nitrocellulose membrane. Immunoblotting was performed using antibodies to ATM (Abcam, Cambridge, England, 1:2000), β-Tubulin (ThermoFisher Scientific, Waltham, USA, 1:2000), c-Myc (Abcam, 1:2000), γH2AX (Novus Biologicals, Centennial, USA, 1:2000), HIF-1α (Cayman Chemical, Ann Arbor, USA, 1:650), HIF-2α (Novus Biologicals, 1:2000) and YY1 (Santa Cruz Biotechnology, Dallas, USA, 1:1000). 5% milk in TBS-T buffer was used for antibody dilution and blocking. Densitometrical quantification of Western blot signals was assessed using ImageJ software (NIH, Bethesda, USA) and relative protein levels were obtained by normalization to Tubulin signal.

Wappler J., Arts M., Röth A., Heeren R.M., Peter Neumann U., Olde Damink S.W., Soons Z, & Cramer T. (2019). Glutamine deprivation counteracts hypoxia-induced chemoresistance. Neoplasia (New York, N.Y.), 22(1), 22-32.

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Comprehensive Molecular Analysis Techniques

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Real-time PCR analysis was performed with primers and probe sets from Applied Biosystems, as described^{49 (link)}. Immunoblots were performed as described previously^{24 (link)}, using the following antibodies: Lck (2752), HK2 (C64G5), p85 (19H8), PDK1 (3062), p-S6 (2F9), p-4EBP1 (236B4), c-Myc (D84C12; all from Cell Signaling Technology), LC3 (NB100-2220; Novus), p110δ (EPR386; Abcam), HIF1α (10006421; Cayman) and β-actin (AC-15; Sigma).

Wei J., Long L., Yang K., Guy C., Shrestha S., Chen Z., Wu C., Vogel P., Neale G., Green D.R, & Chi H. (2016). Autophagy enforces functional integrity of regulatory T cells by coupling environmental cues and metabolic homeostasis. Nature immunology, 17(3), 277-285.

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Protein Expression Analysis Protocol

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Preparation of protein extracts, immunoblots, and fluorescent detection was done as previously described using the Li-Cor Odyssey system (Li-Cor, Lincoln, NE) [34 (link)]. Antibodies used include HIF1α (rabbit polyclonal, Cayman Chemicals), Cyclin D1 (rabbit polyclonal, ThermoScientific), Patched 1 (goat polyclonal, Santa Cruz), Gli1 (rabbit polyclonal, Santa Cruz), Gli3 (goat polyclonal, R and D Systems), N Myc (mouse monoclonal, Millipore), and β-Actin (mouse ascites, Sigma).

Nguyen V., Sabeur K., Maltepe E., Ameri K., Bayraktar O, & Rowitch D.H. (2017). Sonic Hedgehog Agonist Protects Against Complex Neonatal Cerebellar Injury. Cerebellum (London, England), 17(2), 213-227.

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Western Blotting Protein Detection

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Immunoblotting was performed as described previously^{6 (link)}. Primary antibodies were followed by mouse- or rabbit-conjugated horseradish peroxidase (HRP). HRP-conjugated antibodies (anti-mouse or anti-rabbit IgG HRP conjugate, Promega) were detected by enhanced chemiluminescence detection (Thermofisher). This included the following antibodies: Akt (9272, Cell Signaling), Cpt1a (15184-1-AP, Proteintech group), Hif-1α (10006421, Cayman Chemical Company), phosphor-Akt (4060, Cell Signaling), PIK3cγ (ab140310, abcam), phosphor-mTOR (5536, Cell Signaling), PTEN (9552, Cell Signaling), phosphor-p70 S6K (9204, Cell Signaling). Alternatively, primary antibodies were followed by fluorescently labeled anti-mouse or rabbit antibodies (LiCor) and imaged using the Odyssey infrared imaging system (LiCor). This included the following antibodies: Glut1 (ab652, Abcam), hexokinase 2 (2867, Cell Signaling), hexokinase 1 (ab104835, Abcam), cytochrome C (556432, BD Biosciences), β-actin (A5441, Sigma) and phosphor-S6 (4858, Cell Signaling). All primary antibodies were used at a 1:1,000 dilution, with secondary HRP antibodies used at 1:8000. Secondary LiCor antibodies were used at 1:10000 (anti-mouse) or 1:8000 (anti-rabbit).

Gerriets V.A., Kishton R.J., Johnson M.O., Cohen S., Siska P.J., Nichols A.G., Warmoes M.O., de Cubas A.A., MacIver N.J., Locasale J.W., Turka L.A., Wells A.D, & Rathmell J.C. (2016). Foxp3 and Toll-like receptor signaling balance Treg cell anabolic metabolism for suppression. Nature immunology, 17(12), 1459-1466.

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Western Blot Analysis of Cellular Stress Markers

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Cells were homogenized with passive lysis buffer (Promega), normalized for protein content, boiled with SDS loading buffer and separated by SDS-PAGE. Proteins were transferred to PVDF membrane (Whatman) and blotted for CGL (ab151769 Abcam), HIF1α (10006421 Cayman Chemical), p-eIF2α Ser51 (9712S Cell Signaling), total eIF2α(9722S Cell Signaling), ATF4 (11815 Cell Signaling), Actin (13E5 Cell Signaling) and Tubulin (2146S Cell Signaling) and secondarily with HRP-conjugated anti-rabbit antibody (Dako).

Longchamp A., Mirabella T., Arduini A., MacArthur M.R., Das A., Treviño-Villarreal J.H., Hine C., Ben-Sahra I., Knudsen N.H., Brace L.E., Reynolds J., Mejia P., Tao M., Sharma G., Wang R., Corpataux J.M., Haefliger J.A., Ahn K.H., Lee C.H., Manning B.D., Sinclair D.A., Chen C.S., Ozaki C.K, & Mitchell J.R. (2018). Amino acid restriction triggers angiogenesis via GCN2/ATF4 regulation of VEGF and H2S production. Cell, 173(1), 117-129.e14.

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Comprehensive Molecular Analysis Techniques

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