Equal amounts of protein were analyzed in duplicate by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The following monoclonal antibodies were used: anti–phospho-Akt (Ser473, Cell signaling, Danvers, MA), anti–total Akt (Cell signaling), anti-phospho-mTOR (Ser2448, Cell signaling), anti-phospho-mTOR (Thr2446, Millipore, Bedford, MA), anti-total mTOR (Cell signaling), anti-HIF-1α (Cayman Chemical, Ann Arbor, MI), anti-VHL (Cell signaling), anti-phospho-AMPK (Thr172, Cell signaling), anti-total AMPK (Cell signaling) anti-phospho-P70S6K (Thr389, Cell signaling), anti-total P70S6K (Cell signaling), anti-phospho-4E-BP (Thr37/46, Cell signaling), anti-lactate dehydrogenase A (LDHA) (Cell signaling), anti-hexokinase-2 (HK-2, SantaCruz Biotechnology, Dallas, TX), anti-phospho-TSC2 (Ser1387) (Cell signaling), anti-total TSC2 (Cell signaling), and anti-β-actin (Bethyl, Montgomery, TX, US). Immunoreactive proteins were detected by horseradish peroxidase–conjugated secondary antibodies and enhanced using chemiluminescence (ECL) reagents (GE Healthcare Life Sciences, Piscataway, NJ). All immunoblots were performed with triplicate and visualized by LAS image analyzer (Fujifilm, Tokyo, Japan). The band density was quantified using MultiGauge (Fujifilm).
Anti hif 1α
Manufactured by Cayman Chemical
Sourced in United States, United Kingdom
Anti-HIF-1α is a laboratory reagent used to detect and quantify the transcription factor hypoxia-inducible factor-1α (HIF-1α). HIF-1α is a subunit of the heterodimeric HIF-1 complex, which plays a central role in the cellular response to hypoxia. Anti-HIF-1α can be used in various research applications, such as Western blotting, immunohistochemistry, and ELISA, to measure HIF-1α levels in biological samples.
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Lab products found in correlation
Anti akt, by Cell Signaling Technology (3 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
Anti phospho akt, by Cell Signaling Technology (2 mentions)
Immobilon p, by Merck Group (2 mentions)
Anti β actin antibody, by Novus Biologicals (2 mentions)
Fluorescence labeled secondary antibody, by LI COR (2 mentions)
M per lysis buffer, by Thermo Fisher Scientific (2 mentions)
Phospho akt ser473, by Cell Signaling Technology (2 mentions)
Phospho erk thr202 tyr204, by Cell Signaling Technology (2 mentions)
Trans blot, by Bio-Rad (2 mentions)
Anti β tubulin, by Cell Signaling Technology (2 mentions)
Anti hif 2α, by Novus Biologicals (2 mentions)
Anti β actin, by Abcam (2 mentions)
Anti mtor, by Cell Signaling Technology (2 mentions)
Anti α sma, by Abcam (2 mentions)
Anti erk1 2, by Cell Signaling Technology (2 mentions)
Anti gapdh, by Cell Signaling Technology (2 mentions)
Anti β actin, by Merck Group (2 mentions)
Anti pdgfrβ, by Cell Signaling Technology (2 mentions)
Immobilon p membrane, by Merck Group (2 mentions)
24 protocols using anti hif 1α
1
Quantitative Immunoblot Analysis of mTOR Signaling
2
Western Blot Analysis of Cellular Signaling
3
Stabilizing HIF-1α Protein in Spleens
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So that rapid degradation of HIF-1α protein could be avoided, spleens were removed from the animals, and proteins were isolated in a hypoxic workstation (1% O2). To determine whether HIF-1α was stabilized because of a hypoxic environment within the spleen after FV infection, a portion of the spleen was removed from the workstation and incubated with 20.9% O2 for 10 minutes for induction of reoxygenation and, thus, HIF-1α degradation. Western blot analysis was performed as previously described54 (link), and protein was loaded (up to 100 µg per lane). The following polyclonal antibodies were used: anti-HIF-1α (Cayman Chemical, Ann Arbor, MI, USA), anti-MST1 (pro-MST1; Abcam, Cambridge, UK), anti-MST1 (beta-chain, Abcam), and anti-ACTIN (Sigma-Aldrich).
4
Western Blot Analysis of Apoptotic Markers
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Cells, untransfected or transfected with siRNA, were lysed in 70 μL of lysis buffer (50 mM Tris HCl pH 7.5, 0.1% Triton, 5 mM EDTA complemented by Proteases and Phosphatases inhibitors (Chemicon Millipore, Billerica, MA, USA and Sigma Aldrich, Saint-Louis, MO, USA) at 1/100). Western blots were performed as previously described [44 (link),45 (link)] using monoclonal rabbit antibodies, anti-Int6 (1/1000, Abcam, Paris, France), anti-HIF-2α (1/1000, Novus Biologicals, Cambridge, UK), anti-HIF-1α (1/1000, Cayman Chemical, Ann Harbor, MI, USA), anti-Caspase 3/7 (1/1000, Cell Signaling, Danvers, MA, USA), anti-cleaved caspase 3/7 (1/1000, Cell Signaling, Danvers, MA, USA) anti-PARP (1/1000, Cell Signaling, Danvers, MA, USA), anti-Bax (1/1000, Cell Signaling, Danvers, MA, USA), anti-Bcl-XL (1/1000, Cell Signaling, Danvers, MA, USA), anti-Bcl-2 (1/1000, Cell Signaling, Danvers, MA, USA), anti-MEK1 (1/1000, Cell Signaling, Danvers, MA, USA), anti-ERK (1/1000, Cell Signaling, Danvers, MA, USA), anti-phospho ERK (1/1000, Cell Signaling, Danvers, MA, USA), and were normalized using a mouse monoclonal antibody anti-actin (1/10,000, Chemicon Millipore, Billerica, MA, USA) or a rabbit polyclonal antibody anti-β-tubulin (1/1000, Cell Signaling, Danvers, MA, USA). Gel quantification was performed using ImageJ (Windows 1.47, Research Services Branch, NIH, Bethesda, MD, USA).
5
Hypoxic Exposure and HIF-1α Detection
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Hypoxic exposure of PASMC, protein extraction, and Western blot analysis were performed as described (5 (link)). Primary antibodies used were anti–HIF-1α (1:1000 dilution; Cayman Chemical) and anti–β-actin (1:50,000 dilution; Abcam). Specific immune-reactive signals were detected using enhanced chemiluminescence ECL Prime Western Blotting System (GE Healthcare) using a proprietary secondary antibody coupled to horseradish peroxidase (1:5000 dilution; Promega).
6
Tissue Hypoxia Detection in Mice
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In some experiments, animals were i.n. injected with 60 mg/kg of Hypoxyprobe (Hypoxyprobe Inc.) to detect tissue hypoxia. After 2 h, mice were sacrificed, and lungs were collected. The lung of each mouse was homogenized and lysed with radioimmunoprecipitation assay buffer (1% Triton X-100, 280 mM NaCl, 0.1% SDS, 0.2% sodium deoxycholate, 5 mM EGTA, and proteinase inhibitor). Immunoblotting was performed with the following antibodies: anti-HIF1α (Cayman Chemical), anti–β-actin (Santa Cruz), and anti-hypoxyprobe mAb (Hypoxyprobe Inc.).
7
Metformin Effects on Autophagy Signaling
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Cells treated or not with metformin (10mM), were lysed in 70μL of lysis buffer (50mM Tris HCl pH 7.5, 0.1% Triton, 5mM EDTA complemented by protease (Chemicon Millipore) and phosphatase (Sigma-Aldrich) inhibitors. Western blots were performed as previously described using monoclonal rabbit antibodies [20 (link), 22 (link)], anti-LC3b (1/1000, Cell Signaling), anti-Beclin 1 (1/1000, Cell Signaling), anti-p62 (1/1000 Abcam), anti-phospho (T172) AMPK (1/1000, Cell Signaling), anti-AMPK (1/1000, Cell Signaling) anti-phospho (S79) ACC (1/1000, Cell Signaling), anti-ACC (1/1000, Cell Signaling), anti-phospho (S2448) mTOR (1/1000, Cell Signaling), anti-mTOR (1/1000, Cell Signaling), anti-phospho (T389) p70S6 Kinase (1/1000, Cell Signaling), anti-p70S6 Kinase (1/1000, Cell Signaling), anti-phospho (T37/46) 4EBP1 (1/1000, Cell Signaling), anti-phospho (S473) AKT (1/1000, Cell Signaling), anti-phospho (T308) AKT (1/1000, Cell Signaling), anti-AKT (1/1000, Cell Signaling), anti-HIF-1α (1/1000, Cayman Chemical), anti-Redd1/DDIT4 (1/1000, Abcam and Proteintech) and were normalized using a rabbit polyclonal antibody anti-β-tubulin (1/1000, Cell Signaling). Gel quantification was performed using ImageJ (Windows 1.47, Research Services Branch, NIH).
8
Western Blot Analysis of HIF-1α
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Western blot analysis was performed as previously described [45 (link)] with 20 µg protein lysate per line. The following antibodies were used: 1:500 anti-HIF-1α (Cayman Chemical, #10,006,421) and 1:1000 anti-α-Tubulin (Santa Cruz Biotechnology, #sc-8035).
9
Antibody Sources for Protein Analysis
10
Protein Expression Analysis Workflow
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For protein preparation, tissues and cells were homogenized in ice-cold HEN buffer (5 mM EDTA, 250 mM NaCl, 50 mM Hepes pH 7.3, 5 mM DTT) containing 1 mM Na3VO4, 0.2% IGEPAL CA-630 (Sigma) and 1% protease inhibitor cocktail (Sigma). Homogenized samples were centrifuged for 30 min. at high speed in a microcentrifuge, after which protein containing supernatant was collected. Protein concentration was determined using a protein assay kit from Bio-Rad. From each sample, 50 µg of protein was separated by electrophoresis on SDS–polyacrylamide gels and electroblotted onto PVDF membranes. Blots were incubated in blocking solution (5% non-fat milk in PBS, 0.1% Tween-20 [PBS-T]), followed by overnight incubation with the following antibodies: anti-HIF1α (Cayman, CAY-1006421; dilution 1∶500); anti-Glut1 (Millipore, 07-1401; dilution 1∶800); anti-p21WAF1/Cip1 (Santa Cruz Biotechnology Inc., sc397; dilution 1∶200); and anti-β-actin (Abcam, ab6276; dilution 1∶5000). The membranes were then washed with PBS-T and incubated with either a HRP conjugated goat anti-rabbit IgG antibody (Thermo, 31460; dilution 1∶10000) or HRP conjugated sheep anti-mouse IgG antibody (ECL, NA931V; dilution 1∶10000). Antibody detection was performed with an enhanced chemiluminescence reaction (Clarity western ECL substrate; Bio-Rad).
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