ELISA kits were used to determine the concentration of HIF-1α (cat. no. DYC1935-2; R&D Systems, Inc.), HIF-2α (cat. no. DYC1997-2; R&D Systems, Inc.), HIF-3α (Spbio, Wuhan, China) and VEGF (cat. no. MMV00; R&D Systems, Inc.) or interleukin-27 (cat. no. DY2274; R&D Systems, Inc.) in the bacterial supernatants. The indicated sample solutions were added to ELISA well plates and the specific-kit cytokine was bound via the immobilized antibody on the plate. Upon washing off the unbound substances, an enzyme-linked polyclonal antibody that was specific to each cytokine was added to the wells. Upon washing the unbound antibody-enzyme molecules, a substrate solution was added to the wells and the intensity of the color in the reaction developed proportionally to the quantity of specific cytokine bound in the initial step, as indicated in the manufacturer's protocol. The reaction was terminated and the color intensity was determined using a microplate reader at 450 nm.
Hif 2α
Manufactured by R&D Systems
Sourced in United States, United Kingdom
HIF-2α is a protein that plays a key role in the cellular response to hypoxic conditions. It is a subunit of the hypoxia-inducible factor (HIF) transcription complex, which regulates the expression of genes involved in angiogenesis, erythropoiesis, and metabolism. HIF-2α is a sensitive marker of hypoxic conditions and is commonly used in research applications to study the effects of low oxygen environments on cells and tissues.
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Lab products found in correlation
Hif 1α, by R&D Systems (3 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Hif 1α, by BD (2 mentions)
Cd133, by MyBioSource (2 mentions)
Cxcr4, by Proteintech (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Flt 1, by Abcam (1 mentions)
Lamin a c, by Cell Signaling Technology (1 mentions)
Ne per nuclear and cytoplasmic extraction kit, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit igg hrp, by Santa Cruz Biotechnology (1 mentions)
Ecl prime, by GE Healthcare (1 mentions)
Total jak2, by Cell Signaling Technology (1 mentions)
Phospho hsl ser563, by Cell Signaling Technology (1 mentions)
Total stat5, by Cell Signaling Technology (1 mentions)
Hif 1α, by Merck Group (1 mentions)
Glyceraldehyde 3 phosphate dehydrogenase gapdh, by Santa Cruz Biotechnology (1 mentions)
Ecl western blot detection reagent, by GE Healthcare (1 mentions)
Hif 1α, by Novus Biologicals (1 mentions)
β actin, by Merck Group (1 mentions)
Ab37067, by Abcam (1 mentions)
10 protocols using hif 2α
1
Quantifying Cytokines in Bacterial Supernatants
2
Normoxic and Hypoxic Protein Extraction
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Cytoplasmic and nuclear extracts under normoxic or hypoxic conditions were prepared using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific, Rockford, IL, USA). Soluble fractions were prepared by the sampling buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 1x Protease Inhibitor Cocktail, 1x Phosphatase Inhibitor Cocktail)41 (link). Ten micrograms of nuclear protein were separated on 4–8% Tris-Acetate gels (Thermo Scientific), and then transferred to polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). After blocking in 5% dry milk in Tris-buffered saline with Tween 20, the membrane was incubated with primary antibodies followed by incubation with horseradish peroxidase-conjugated secondary antibodies. Detection was performed using ECL Prime (GE Healthcare, Logan, UT, USA) according to the manufacturer's instructions. The following antibodies were used: HIF-1α (NB100-449, Novus Biologicals, Littleton, CO, USA), HIF-2α (AF2997, R&D systems, NE, USA), Lamin A/C (2032S, Cell Signaling Technology, Danvers, MA, USA), goat anti-rabbit IgG-HRP (sc-2004, Santa Cruz Biotechnology, Santa Cruz, CA, USA), Flt-1 (ab9540, Abcam, Camgridge, UK), CXCR4 (66042, Proteintech, Illinois, USA). The non-cropped blots of the representative images are shown in Supplementary Fig. 10 . This experiment was successfully repeated three times.
3
Western Blot Analysis of Cell Signaling
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Tissue or cells were lysed in ice-cold RIPA lysis buffer supplemented with protease inhibitor and phosphatase inhibitor cocktails. Western blots were probed with the following antibodies: total JAK2, total STAT5, phospho-JAK2 (Tyr1007/1008) phospho-STAT5 (Tyr694) , ATGL , HSL and phospho-HSL (Ser563) purchased from Cell Signaling Technology (Danvers, MA, USA); EPOR (R&D Systems, Minneapolis, MN, USA); HIF-1α (Sigma-Aldrich, Saint Louis, MO, USA); HIF-2α (R&D Systems) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology, Santa Cruz, CA, USA).
4
Hypoxic Regulator Protein Detection
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Proteins (25 µg) were run on a SDS-polyacrylamide gel and then transferred onto a nitrocellulose membrane. The TATA binding protein (TBP) or β-actin were used as loading control. The membrane was incubated overnight at 4°C with primary antibodies against HIF-1α (0.2%; Novus Biologicals, Cambridge, UK), HIF-2α (0.5%; R&D Systems, Wiesbaden, Germany), PHD3 (0.1%; Novus Biologicals), β-actin (0.2%; Sigma-Aldrich) or TBP (0.1%; Abcam, Cambridge, UK) followed by incubation with appropriate HRP-conjugated secondary antibodies (0.02%; Dianova; Thermo Fisher Scientific, Bonn, Germany) for 1 h. All antibodies were diluted in blocking buffer consisting of 5% nonfat dry milk in TBST (0.1% Tween-20 in TBS). ECL Western blot detection reagents (GE Healthcare, Freiburg, Germany) were used for protein detection.
5
Western Blot Analysis of HIF-1α and Related Proteins
6
Hypoxia Modulates Glioma Stem Cell Markers
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New spheres formed by single glioma cells and CD133-CD15- cells cultured in 21% O2 (control group) or 1% O2 for 72 h were evaluated for HIF1α, HIF2α, SOX-2, CD133 and CD15 expression by western blotting. Total protein was prepared using prechilled RIPA buffer (Beyotime Biotechnology, China), and the isolated proteins were subjected to SDS-PAGE, followed by transfer to nitrocellulose membranes. Then, the membranes were blocked with 5% nonfat milk and incubated with primary antibodies against HIF1α (1:1,000, R&D Systems, USA), HIF2α (1:1,000, R&D Systems, USA), SOX-2 (1:1,000, R&D Systems, USA), CD133 (1:1,000, MyBiosource, USA) and CD15 (1:1,000, R&D Systems, USA) at 4°C overnight. HRP-labeled secondary antibodies (Beyotime Biotechnology, China) were added to the membranes and incubated at 37°C for 1 h. Enhanced chemiluminescence was utilized for visualization.
7
Immunohistochemical Profiling of Brain Tumors
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HIF1α, HIF2α, Sox2, CD133 and CD15 expression in tumor or normal tissues obtained from mouse brains was detected by IHC. Briefly, tissue samples were fixed in formalin and embedded in paraffin, followed by dewaxing in xylene, rinsing in a graded ethanol series and rehydration in double-distilled water. The slides were washed with PBS for 3 min, immunostained using primary antibodies against HIF1α (1:100, R&D Systems, USA), HIF2α (1:100, R&D Systems, USA), SOX-2 (1:100, R&D Systems, USA), CD133 (1:150, MyBiosource, USA) and CD15 (1:100, R&D Systems, USA), and incubated at 4°C overnight. The slides were washed with PBS buffer again and covered with an HRP-labeled polymer for 2 h. Then, the tissue samples were covered with a DAB chromogen solution and incubated for ~1 min to allow staining reactions to occur, and images were acquired.
8
Curcumin-based Anticancer Therapy Assessment
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Curcumin (Exir-nano-sina) was prepared using dimethyl sulfoxide (DMSO) and stored at -20 °C. RPMI-1640, FBS (fetal bovine serum). Penicillin-streptomycin was purchased from Biosera (UckfieldEast Sussex, UK). 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and DMSO were purchased from Sigma (St. Louis, MO). Anti HIF-1α, ARNT and HIF-2α antibodies were provided by R&D Systems Inc. Other antibodies (anti-CD 44 -conjugated microbeads, CD 24 -conjugated biotin and antibiotin-CD 24 ) were purchased from Miltenyi Biotec. Annexin V-FITC and propidium iodide (PI) were purchased from eBioscience (San Diego, CA).
9
Curcumin Modulates Hypoxic Signaling
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The CS-LCs (4 × 10 5 cells/ml/well) were seeded in six-well plates and treated with curcumin under hypoxic and normoxic conditions. After 24 h, the cells were lysed with lysis buffer to extract the total protein [14] . Equal amounts of protein were separated by 10 % SDS-PAGE and transferred to a PVDF membrane (Roche, Germany). Subsequently, the membrane was blocked with 5 % non-fat dry milk in TBS-T (0.05 % Tween-20) for two hours at room temperature and blotted for primary antibodies (HIF-1α: 1:2000; HIF-2α: 1:2000; ARNT: 1:1000 R&D Systems and β-Actin 1:5000 Santa Cruz Biotech) overnight at 4 °C. After washing three times with TBS-T, the membranes were incubated with horseradish peroxidase (HRP)-conju-gated secondary antibody at 1: 5000 for 60 min at room temperature and developed using enhanced chemical luminescence detection system (ECL) (Roche, Germany). Bands were analyzed using the Image J software and normalized to corresponding β-Actin band intensity.
10
Western Blot Analysis of HIF Proteins
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Protein was extracted from ECFCs using ice-cold RIPA containing protease and phosphatase inhibitors as described previously. Twenty micrograms of protein was resolved by electrophoresis through 10% SDS polyacrylamide gel and electro-transferred onto PVDF membrane (Millipore). The membranes were blocked for 1 h at RT with 3% (w/v) non-fat dry milk in Tris-buffered saline with 0.1% Tween-20 (TBS-T). After blocking, the membranes were incubated overnight at 4 °C with primary antibodies reactive to HIF-1α (#610958; BD Biosciences) and HIF-2α (R&D Systems). After three washes with TBS-T, the membranes were incubated for 1 h with their respective HRP-conjugated secondary antibodies (Santa Cruz). Three TBS-T washes were carried out before the protein bands were detected using ECL (Merck Millipore). Membranes were re-probed with β-actin (Cell Signaling Technology) as a housekeeping gene to ensure equal loading.
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