Soybean lecithin (S100) was provided by Lipoid GmbH (Ludwigshafen, Germany). Folic acid, oleic acid, cholesterol, chitosan (molecular weight ~110,000–150,000), N-hydroxysuccinimide (NHS), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), and cobalt chloride were purchased from Sigma-Aldrich (St Louis, MO, USA). HIF-1α siRNA (human), control HIF-1α siRNA (fluorescein isothiocyanate [FITC] conjugate)-A, anti-HIF-1α antibody, goat anti-rabbit IgG-HRP, and GAPDH antibody were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Deionized water was prepared by a water purifying system (Woter, Chengdu, People’s Republic of China). All other chemicals were of analytical grade and used as received. The protocol of cell experiments was reviewed and approved by the Medical Ethics Committee of Shanghai Dermatology Hospital.
Anti hif 1α antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The Anti-HIF-1α antibody is a laboratory tool used to detect and study the expression of the Hypoxia Inducible Factor-1α (HIF-1α) protein. HIF-1α is a transcription factor that plays a central role in the cellular response to hypoxic conditions. This antibody can be used in various immunodetection techniques, such as Western blotting, immunohistochemistry, and immunofluorescence, to analyze the levels and localization of HIF-1α in biological samples.
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Lab products found in correlation
Protein a g plus agarose, by Santa Cruz Biotechnology (2 mentions)
Soybean lecithin s100, by Lipoid (1 mentions)
Chitosan, by Merck Group (1 mentions)
Oleic acid, by Merck Group (1 mentions)
Gapdh antibody, by Santa Cruz Biotechnology (1 mentions)
Cholesterol, by Merck Group (1 mentions)
Folic acid, by Merck Group (1 mentions)
Cobalt chloride, by Merck Group (1 mentions)
Goat anti rabbit igg hrp, by Santa Cruz Biotechnology (1 mentions)
Immobilon p membrane, by Merck Group (1 mentions)
Enhanced chemiluminescence plus kit, by Cytiva (1 mentions)
Horseradish peroxidase conjugated anti rabbit antibody, by Santa Cruz Biotechnology (1 mentions)
Cocl2, by Merck Group (1 mentions)
Anti β actin antibody, by Santa Cruz Biotechnology (1 mentions)
Anti notch1 antibody, by Santa Cruz Biotechnology (1 mentions)
N n 3 5 difluorophenacetyl l alanyl s phenylglycine t butyl ester dapt, by Santa Cruz Biotechnology (1 mentions)
Anti n cadherin antibody, by Santa Cruz Biotechnology (1 mentions)
Anti snail antibody, by Santa Cruz Biotechnology (1 mentions)
Anti e cadherin antibody, by Santa Cruz Biotechnology (1 mentions)
Hrp linked anti rabbit and anti mouse igg, by Cell Signaling Technology (1 mentions)
14 protocols using anti hif 1α antibody
1
Soybean Lecithin-based Nanoparticles for HIF-1α Silencing
2
Immunofluorescence Assay for HIF-1α
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Cultured cells were treated with UA for 24 h, and then fixed with cool methanol and washed with PBS. The fixed cells were permeabilized with 0.5% Triton-100 and blocked with 5% BSA, followed by incubation with an anti-HIF-1α antibody (Santa Cruz, CA, USA) for 4 hour. After washing, cells were incubated with anti-mouse FITC secondary antibody for 30 minutes, followed by incubating cells with DAPI for 10 minutes at room temperature.
3
Western Blot Analysis of HIF-1α
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Total proteins were electrophoresed on 8%-12% SDS/polyacrylamide gels, transferred to Immobilon-P membranes (Millipore, Bedford, MA, USA), and blocked with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween-20 (TTBS) for 60 min. The membranes were then sequentially incubated overnight with anti-HIF-1α antibody (Santa Cruz Biotechnology, 1:1,000) or anti-β-tubulin antibody (1:3,000). The membranes were washed with TTBS and incubated with horseradish peroxidase-conjugated secondary antibody (1:5,000) for 1 hr. The blots were visualized using an Enhanced Chemiluminescence Plus Kit (Amersham Biosciences, Piscataway, NJ, USA).
4
Evaluating EMT Markers in Hypoxia
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Anti-E-cadherin antibody, anti-N-cadherin antibody, anti-Snail antibody, anti-HIF-1α antibody, anti-Notch1 antibody, anti-β-actin antibody and horseradish peroxidase-conjugated anti-rabbit antibody, and N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) were purchased from Santa Cruz Biotechnology, Inc., (Dallas, TX, USA). CoCl2 was purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany).
5
Immunoprecipitation of HIF-1α in HCT-116 cells
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HCT-116 cells were lysed in RIPA buffer (50 mM Tris-HCl pH 8.0, 250 mM NaCl, 0.1% SDS, 1% NP-40, 1% sodium deoxycholate, 5 mM EDTA, and 1 mM DTT) supplemented with protease inhibitors. The samples (800 µg) were precleared with 30 µL of Protein A/G PLUS-Agarose (Santa Cruz Biotechnology) for 2 h at 4°C. The anti-HIF-1α antibody (rabbit polyclonal antibody from Santa Cruz Biotechnology) was added to lysate and incubated overnight at 4°C and then 30 µL of Protein A/G PLUS-Agarose were added to the lysate. After 1 h at 4°C the proteins were recovered in Laemmli buffer, resolved on 8% denaturing gel and then subjected to immunoblotting.
6
Antibodies and Inhibitors for Cellular Metabolism
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Monoclonal antibodies against mTOR (2972S), LC3 (12741S), p62 (5114S), PDH (3205S), GAPDH (5174S), PFK (8164S), LDH (2012S), Akt (9272S), p‐Akt (193H12) and β‐actin (4967S) were purchased from Cell Signaling Technology (Danvers, MA, USA), while those against GLUT1 and p‐mTOR were from Invitrogen (Carlsbad, CA, USA; MA5‐31960 and12‐9718‐41, respectively), and those against IDH from Sigma‐Aldrich (HPA007831; St. Louis, MO, USA). Anti HIF‐1α antibody was purchased from Santa Cruz Biotechnology (Dallas, TX, USA; sc‐13 515). Secondary antibodies were HRP‐linked anti‐rabbit and anti‐mouse IgG from Cell Signaling Technology (7074S and 7076S respectively, Danvers, MA, USA). Inhibitors VPC‐23019 and rapamycin were procured from Cayman Chemical Company (Ann Arbor, MI, USA), while JTE‐013 was purchased from Sigma‐Aldrich. Akt inhibitor (Aktin‐1/2), Akt1/2 kinase inhibitor was obtained from Abcam (Cambridge, UK; ab142088). Glucose‐6‐phosphate (G6P) assay kit for enzymatic determination of G6P was purchased from Merck (Darmstadt, Germany; MAK014). FTY‐720 was from Cayman Chemical Company while MTT Assay Kit was from Abcam (ab197010).
7
HIF-1α Interaction with ERβ2
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2μl in vitro translated HIF-1α protein was incubated with 25 μl of bacterial lysate containing His-ERβ2 (LBD) overnight, and complexes were adsorbed onto nickel agarose beads for 2 h. Beads were washed three times with ice-cold NETN (20 mM Tris (pH = 8.0), 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40). Complexes were boiled with 20 μl of 2× sample buffer, subjected to SDS-PAGE and transferred to nitrocellulose membrane for Western analyses. The blot was probed with anti-HIF-1α antibody (Santa Cruz). Primary antibodies were at 1:1000 dilution, and secondary antibody at 1:10,000.
8
Quantifying Hypoxia-Induced Protein Levels
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Cells in the both hypoxia and normoxia groups were collected from the wells and their protein contents were detected using an extraction Kit (Santa Cruz, USA) following the manufacturer’s protocol. Total protein concentration was measured using a Nanodrop (Thermo-Scientific, USA). Samples were prepared for western blotting by adding loading buffer to each sample. Proteins were electrophoresed on 12% SDS-polyacrylamide gel and transferred to PVDF membranes. The membranes were blocked by incubating with 0.3 g bovine serum albumin in 10 ml of washing buffer at 4ºC overnight. Membranes were then washed three times with PBS for 10 min. Then, the membranes were incubated with anti-HIF-1α antibody (dilution: 1:500; Santa-Cruz) for 4 hrs at 4ºC. The membranes were then washed three times for 10 min each and incubated with the secondary antibody for 2 hrs. Roche ECL kit and semi-dry X-ray were used for imaging of immunoreactive protein bands.
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Cells in the both hypoxia and normoxia groups were collected from the wells and their protein contents were detected using an extraction Kit (Santa Cruz, USA) following the manufacturer’s protocol. Total protein concentration was measured using a Nanodrop (Thermo-Scientific, USA). Samples were prepared for western blotting by adding loading buffer to each sample. Proteins were electrophoresed on 12% SDS-polyacrylamide gel and transferred to PVDF membranes. The membranes were blocked by incubating with 0.3 g bovine serum albumin in 10 ml of washing buffer at 4ºC overnight. Membranes were then washed three times with PBS for 10 min. Then, the membranes were incubated with anti-HIF-1α antibody (dilution: 1:500; Santa-Cruz) for 4 hrs at 4ºC. The membranes were then washed three times for 10 min each and incubated with the secondary antibody for 2 hrs. Roche ECL kit and semi-dry X-ray were used for imaging of immunoreactive protein bands.
9
ChIP Assay for HIF1α Binding
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ChIP assays were carried out with cells fixed with 1% formaldehyde using a standard protocol. Immunoprecipitation with anti-HIF1α antibody (Santa Cruz Biotechnology) overnight at 4 °C. For each reaction, 1% of the sample was taken and pre-immune IgG was used as a negative control. Antibody and Chromatin complex were immunoprecipitated by protein A/G agarose beads with salmon sperm DNA (50% Slurry). Eluted DNA-Protein complexes were reversed with 5 M NaCl at 65 °C overnight. Immunoprecipitated DNA was purified and amplified by PCR of HRE binding regions of human or mouse (as described in figures) EPO and VEGF promoters [[39] (link), [40] (link), [41] (link)]. Data were presented as fold enrichment by subtracting the signal with no antibody and expressed as fold increase over the control sample.
10
Quantitative Analysis of PD-L1 and HIF-1α in Liver Cancer
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Construction of liver cancer tissue microarrays as well as immunostaining was performed according to standard protocols described elsewhere [6] (link). The following primary antibodies were used: anti–PD-L1 (mouse monoclonal antibody, clone 2B11D11, Proteintech, Chicago, IL) and anti–HIF-1α antibody (mouse monoclonal antibody, clone H1alpha67, Santa Cruz Biotechnology, CA). PD-L1 and HIF-1α expressions were assessed in neoplastic tumor cells. The semiquantitative analytical criteria of the PD-L1 and HIF-1α expression levels have been previously described in detail [21] (link), [29] (link): cytoplasmic/membranous PD-L1 expression according to the intensity of the staining (0, negative; 1, very weak; 2, moderate; and 3, strong expression) [30] (link) and cytoplasmic and nuclear HIF-1α expression according to intensity and extensity of the staining (0, no staining; 1, nuclear staining in less than 10% of cells and/or with weak cytoplasmic staining; 2, nuclear staining in 10%-50% of cells and/or with distinct cytoplasmic staining; and 3, nuclear staining in more than 50% of cells and/or with strong cytoplasmic staining) [31] (link); 0 and 1 were defined as low-expression group, whereas 2 and 3 were defined as high-expression group.
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