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Hif 1α

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HIF-1α is a protein that plays a crucial role in the cellular response to low oxygen levels (hypoxia). It functions as a transcription factor, regulating the expression of genes involved in processes such as angiogenesis, erythropoiesis, and glucose metabolism.

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Lab products found in correlation

Hif 2α, by GeneTex (2 mentions) Vimentin, by Cell Signaling Technology (2 mentions) Hrp conjugated secondary antibody, by Bio-Rad (2 mentions) β actin, by Cell Signaling Technology (2 mentions) Histone h1, by Santa Cruz Biotechnology (1 mentions) Ph2ax, by Merck Group (1 mentions) Laminin b, by Abcam (1 mentions) Parp 1, by Enzo Life Sciences (1 mentions) Myc tag, by Cell Signaling Technology (1 mentions) Ecl system, by Cytiva (1 mentions) Hrp conjugated secondary igg, by Merck Group (1 mentions) Phosphor p70s6k, by Cell Signaling Technology (1 mentions) Phospho ampk, by Cell Signaling Technology (1 mentions) Ecl detection system, by Thermo Fisher Scientific (1 mentions) Quantity one software, by Bio-Rad (1 mentions) Chemidoc, by Bio-Rad (1 mentions) Enhanced chemiluminescence technique, by PerkinElmer (1 mentions) Ripa cell lysis buffer, by Cell Signaling Technology (1 mentions) Mouse anti hif 1α, by Cell Signaling Technology (1 mentions) β actin, by Merck Group (1 mentions)

6 protocols using hif 1α

1

Western Blot Analysis of Cellular Proteins

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For Western Blot analysis, cells were plated in six-well plates at a density of 2.5 × 105, lysed using TRE buffer and then the whole cell extract was sonicated, resuspended, and boiled for 5 min in modified Laemli charge buffer (250 mM Tris-HCl (pH 75), glycerol 20%, SDS 10%, 1,4 M of mercaptoethanol and 1% blue bromophenol). Samples were transferred to a nitrocellulose membrane through wet transfer (Amersham Biosciences). Proteins were then visualized using the ECL system (Amersham Biosciences) after using specific antibodies for: HIF-1α (Bethyl), GST antibodies (Bethyl), Tubulin (Sigma-Aldrich), Actin (Sigma-Aldrich), Myc-tag (Cell Signaling Technology), Poly(ADP-ribose) (Trevigen), PARP-1 (Enzo), RPA (Cell Signaling), p-RPA (Bethyl), P53 (Santa Cruz), p-P53 (Millipore), H2AX (Millipore), p-H2AX (Millipore), Histone H1 (Santa Cruz), Laminin B (Abcam).
Martí J.M., Garcia-Diaz A., Delgado-Bellido D., O'Valle F., González-Flores A., Carlevaris O., Rodríguez-Vargas J.M., Amé J.C., Dantzer F., King G.L., Dziedzic K., Berra E., de Álava E., Amaral A.T., Hammond E.M, & Oliver F.J. (2021). Selective modulation by PARP-1 of HIF-1α-recruitment to chromatin during hypoxia is required for tumor adaptation to hypoxic conditions. Redox Biology, 41, 101885.
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2

Hypoxia-induced Protein Expression Analysis

Cited 1 time
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Cell lysates were collected 48 h after seeding the cells at 0.5 × 105/well in 24-well plates under early stage conditions (5% CO2, ambient O2, 1000 mg/ml glucose) or advanced stage conditions (20% CO2, 1% O2, no glucose, 5 mM lactate). For generating chemical hypoxia as a positive control, HeLa cells were treated with 100 μM CoCl2 for 48 h before lysing. The protein concentrations were determined using the Pierce BCA assay, and 15 μg of protein was loaded per lane on 8% SDS PAGE. The separated proteins were then transferred to a PVDF membrane, blocked with 5% nonfat milk, probed with antibodies to asTF (rabbit monoclonal RabMab1) 19 (link), vimentin (rabbit mAb, Cell Signaling), β-actin (rabbit, Cell Signaling), HIF-1α (rabbit pAb, Bethyl Laboratories), HIF-2α (rabbit pAb, GeneTex) CAIX (mouse mAb, GeneTex), or CAXII (goat pAb, Acris), followed by probing with the corresponding HRP-conjugated secondary antibodies (Bio-Rad) and developed using ECL and H2O2.
Ramchandani D., Unruh D., Lewis C.S., Bogdanov V.Y, & Weber G.F. (2016). Activation of Carbonic Anhydrase IX by Alternatively Spliced Tissue Factor Under Late-Stage Tumor Conditions. Laboratory investigation; a journal of technical methods and pathology, 96(12), 1234-1245.
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3

Western Blot Analysis of Autophagy Markers

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Cells or isolated mitochondria were lysed in the RIPA buffer containing Na3VO4 (1 mM) and protease inhibitors (1 μg/ml). The cell lysate proteins were separated using SDS-PAGE and transferred onto nitrocellulose membranes (Protran). After blocking with 5% non-fat milk in PBS-Tween (0.1%), the membranes were incubated with antibodies against IRS1, HIF1α (both Bethyl), BECN1, SQSTM1/p62, LC3B, phosphor-p70S6K, phospho-AMPK, AMPK, phospho-ULK1 (Ser-555), phospho-ULK1 (Ser-757), and ULK1 (all Cell Signaling), phospho-mTOR (p-mTOR, Ser-2448), mTOR, PARK2 (all ThermoFisher Scientific), and GLUT-4, cytochrome c and VDAC1 (all Santa Cruz). After incubation with the HRP-conjugated secondary IgG (Sigma), blots were developed using the ECL detection system (Pierce Biotechnology). The band intensities were visualized and quantified with ChemiDoc using the Quantity One software (BioRad Laboratories).
Tomasetti M., Monaco F., Manzella N., Rohlena J., Rohlenova K., Staffolani S., Gaetani S., Ciarapica V., Amati M., Bracci M., Valentino M., Goodwin J., Nguyen M., Truksa J., Sobol M., Hozak P., Dong L.F., Santarelli L, & Neuzil J. (2016). MicroRNA-126 induces autophagy by altering cell metabolism in malignant mesothelioma. Oncotarget, 7(24), 36338-36352.
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4

Hypoxia-induced Protein Expression Analysis

Cited 1 time
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Cell lysates were collected 48 h after seeding the cells at 0.5 × 105/well in 24-well plates under early stage conditions (5% CO2, ambient O2, 1000 mg/ml glucose) or advanced stage conditions (20% CO2, 1% O2, no glucose, 5 mM lactate). For generating chemical hypoxia as a positive control, HeLa cells were treated with 100 μM CoCl2 for 48 h before lysing. The protein concentrations were determined using the Pierce BCA assay, and 15 μg of protein was loaded per lane on 8% SDS PAGE. The separated proteins were then transferred to a PVDF membrane, blocked with 5% nonfat milk, probed with antibodies to asTF (rabbit monoclonal RabMab1) 19 (link), vimentin (rabbit mAb, Cell Signaling), β-actin (rabbit, Cell Signaling), HIF-1α (rabbit pAb, Bethyl Laboratories), HIF-2α (rabbit pAb, GeneTex) CAIX (mouse mAb, GeneTex), or CAXII (goat pAb, Acris), followed by probing with the corresponding HRP-conjugated secondary antibodies (Bio-Rad) and developed using ECL and H2O2.
Ramchandani D., Unruh D., Lewis C.S., Bogdanov V.Y, & Weber G.F. (2016). Activation of Carbonic Anhydrase IX by Alternatively Spliced Tissue Factor Under Late-Stage Tumor Conditions. Laboratory investigation; a journal of technical methods and pathology, 96(12), 1234-1245.
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5

HIF-1α Protein Expression Analysis

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Total cell lysate was extracted from HCC cells using RIPA cell lysis buffer (Cell signaling, Danver, MA, USA) according to the manufacturer’s instructions. HIF-1α was bounded with mouse anti-HIF-1α (Cell signaling, Danver, MA, USA) using IgG magnetic beads (Novex, Oslo, Norway), according to the manufacturer’s instructions. The dimerized proteins were detected according to western blot, and blotted with the other target-mouse monoclonal antibodies-HIF-1α (Bethyl, Montgomery, TX, USA) specific for the proteins of interest. The blots were developed using the enhanced chemiluminescence technique (PerkinElmer, Boston, MA, USA) according to the manufacturer’s instructions, and the level of expression of each protein was quantified and compared.
Choi S.H., Chung A.R., Kang W., Park J.Y., Lee M.S., Hwang S.W., Kim D.Y., Kim S.U., Ahn S.H., Kim S, & Han K.H. (2014). Silencing of Hypoxia-Inducible Factor-1β Induces Anti-Tumor Effects in Hepatoma Cell Lines under Tumor Hypoxia. PLoS ONE, 9(7), e103304.
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6

Macrophage response to lactate treatment

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A total of 2 × 106 BMDMs were plated in 60-mm tissue culture plates, grown overnight, serum-starved for 24 hours, and treated with 10 mM lactate for 4 and 6 hours. As an untreated control, macrophages were also incubated in starvation medium alone for 6 hours. Macrophages were harvested, lysed, separated on SDS-PAGE, and probed for HIF-1α (Bethyl Laboratories, Montgomery, TX) and β-actin (Sigma) as a loading control. Band intensities were quantified by densitometric analysis using ImageJ software, and relative gel densities were determined by normalizing to β-actin as described previously [27] (link).
Lee S., Roh H.S., Song S.S., Shin J., Lee J., Bhang D.H., Kim B.G., Um S.H., Jeong H.S, & Baek K.H. (2020). Loss of S6K1 But Not S6K2 in the Tumor Microenvironment Suppresses Tumor Growth by Attenuating Tumor Angiogenesis. Translational Oncology, 13(4), 100767.
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