Mouse anti hif1α

Manufactured by BD

Sourced in United States

Mouse anti-HIF1α is a monoclonal antibody that specifically recognizes the hypoxia-inducible factor 1-alpha (HIF1α) protein. HIF1α is a transcription factor that plays a crucial role in the cellular response to low oxygen conditions (hypoxia).

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13 protocols using mouse anti hif1α

Hypoxia Markers in Laryngeal Carcinoma

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Laryngeal tumor biopsies from 58 patients with advanced laryngeal squamous cell carcinoma were used in this study. The patients participated in a phase III randomized trial where they received accelerated radiotherapy with or without carbogen gas and nicotinamide.⁶ (link) 79 patients participated in a multicenter translational side study and received PIMO intravenously two  hours before biopsy. In this present study, the tissue of 58 patients who were included in the RadboudUMC, Nijmegen, the Netherlands (single-center) was used.
Consecutive sections were cut from each tissue block and immunohistochemically stained for two hypoxic markers, HIF-1α, and PIMO. The staining procedure was done as previously described.³ (link) For PIMO, we used the primary antibody Mouse-antiPIMO (Lot# 9.7.11, HydroxyProbe, Massachusets, USA). For the HIF-1α IHC, the Novolink kit (Leica Biosystems, Rijswijk, the Netherlands) was used with the primary antibody Mouse-anti-HIF-1α (BD Biosciences, cat# 610959, lot 4 073 775).

Smits H.J., Swartz J.E., Philippens M.E., de Bree R., Kaanders J.H., Koppes S.A., Breimer G.E, & Willems S.M. (2023). Validation of automated positive cell and region detection of immunohistochemically stained laryngeal tumor tissue using digital image analysis. Journal of Pathology Informatics, 14, 100198.

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Lysolipid Signaling Pathway Analysis

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LPA (18:1) was purchased from Avanti Polar Lipids (Alabaster, AL). LPA was used at the final concentration of 1 μM in PBS containing 0.1% fatty acid-free bovine serum albumin (BSA) unless otherwise specified. The same volume of PBS containing 0.1% BSA was used as a control. The following antibodies were purchased: mouse anti-HIF1α (BD Biosciences, Franklin Lakes, NJ); rabbit anti-MIF, mouse anti-FLAG, and mouse anti-actin (Sigma-Aldrich, St. Louis, MO); mouse anti-HA (Covance, Princeton, NJ). Human recombinant MIF (rMIF) was from R&D System (Minneapolis, MN). MIF antagonist, (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1) was obtained from EMD Millipore (Billerica, MA). All other chemicals and reagents were purchased from Sigma.

No Y.R., Lee S.J., Kumar A, & Yun C.C. (2015). HIF1α-Induced by Lysophosphatidic Acid Is Stabilized via Interaction with MIF and CSN5. PLoS ONE, 10(9), e0137513.

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Western Blot Analysis of Metabolic Regulators

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Cell lysis, protein sample preparation and Western blot were carried out as previously described [27 (link)]. Briefly, primary antibodies mouse anti-MCT1 (1:500, sc-365501, Santa Cruz Biotechnology), rabbit anti-MCT4 (1:500, sc-50329, Santa Cruz Biotechnology), mouse anti-CD147 (1:500, sc-71038, Santa Cruz Biotechnology), mouse anti-HIF-1α (1:500, 610958, BD Biosciences) and goat anti-actin (1:500, sc-1616, Santa Cruz Biotechnology) were used. Membranes were then incubated with the adequate secondary antibodies coupled to horseradish peroxidase (Santa Cruz Biotechnology) and bound antibodies were visualised by chemiluminescence (Supersignal West Femto kit, Pierce, Rockford, IL, USA). Protein quantification was performed using ImageJ Software (version 1.41).

Morais-Santos F., Granja S., Miranda-Gonçalves V., Moreira A.H., Queirós S., Vilaça J.L., Schmitt F.C., Longatto-Filho A., Paredes J., Baltazar F, & Pinheiro C. (2015). Targeting lactate transport suppresses in vivo breast tumour growth. Oncotarget, 6(22), 19177-19189.

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Antibody Use in Hypoxia Signaling

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The primary antibodies used were as follows: mouse anti‐HIF1α (BD Biosciences), rabbit anti‐HIF1α (D2U3T), rabbit anti‐NDRG1 (D8G9), rabbit anti‐BNIP3 (D7U1T), rabbit anti‐ATF4 (D4B8), mouse anti‐LAMP1 (D4O1S) (Cell Signaling Technology), rabbit anti‐ERK2 (C14), mouse anti‐GAPDH (6C5), goat anti‐LDHA (N14) (Santa Cruz Biotechnologies), mouse anti‐MITF (C5) (Millipore), mouse anti‐MITF (D5) (Dako), rabbit anti‐MITF (HPA003259), rabbit anti‐SDHB (HPA002868) (Cambridge Biosciences). Rabbit anti‐HIF1α and anti‐HIF2α used in ChIP‐seq experiments were provided by the Ratcliffe laboratory, rabbit anti‐HIF1β (Novus Biologicals), mouse anti‐FLAG (M2) (Sigma), and mouse anti‐HA (12CA5) (Roche). Alexa Fluor‐conjugated secondary antibodies were obtained from Invitrogen.

Louphrasitthiphol P., Ledaki I., Chauhan J., Falletta P., Siddaway R., Buffa F.M., Mole D.R., Soga T, & Goding C.R. (2019). MITF controls the TCA cycle to modulate the melanoma hypoxia response. Pigment Cell & Melanoma Research, 32(6), 792-808.

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Western Blot Analysis of HIF and NF-kB

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Nuclear extracts were prepared by a nuclear extract kit (Active Motif). A total of 5–20 µg proteins were resolved on Tris-HCl polyacrylamide gels and electrophoretically transferred to polyvinylidene difluoride membranes (Invitrogen). Membranes were blocked in PBS-fat free milk 5% (Bio-Rad Laboratories) for 1 h, probed with the appropriate primary antibody overnight at 4°C and subsequently incubated with peroxidase conjugated secondary antibodies for 1 h at room temperature. Immunocomplexes were visualized using an enhanced chemiluminescent kit (EuroClone). Digital images of the resulting bands were quantified by the Quantity One software package (Bio-Rad Laboratories) and expressed as arbitrary densitometric units.
The following primary and secondary antibodies were used: mouse anti-HIF1α (1∶500, BD Bioscience); mouse anti-HIF2α and rabbit anti-p50 (1∶500, 1∶1000, Novus Biologicals); rabbit anti-HIF3α (1∶1000, Aviva Biological Systems); mouse anti-p65 (1∶2000, Santa Cruz Biotechnology); rabbit anti phospho-NF-kB p65 (Ser 276) (1∶1000, Cell Signaling); mouse anti β-actin (1∶10000, Sigma Aldrich); horseradish peroxidase conjugated anti-mouse and anti-rabbit (1∶2000, Bio-Rad Laboratories).

Ravenna L., Principessa L., Verdina A., Salvatori L., Russo M.A, & Petrangeli E. (2014). Distinct Phenotypes of Human Prostate Cancer Cells Associate with Different Adaptation to Hypoxia and Pro-Inflammatory Gene Expression. PLoS ONE, 9(5), e96250.

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Western Blot Analysis of HIF-1α Expression

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Mouse anti-HIF-1α (BD Biosciences, Le Pont de Claix, France) was used as primary antibody. Proteins were visualized by an enhanced chemiluminescence detection system (GE Healthcare, Vélizy-Villacoublay, France) using anti-mouse horseradish peroxidase-conjugated IgG (Bio-Rad, Hercules). Equal loading of protein was confirmed by probing the blots with anti-α-tubulin or anti-ß-actin antibodies (Sigma-Aldrich). Densitometry quantitation was determined with Image J software 1.53i using the area under the peak method (NIH, Bethesda, MD, USA).

Gomez-Brouchet A., Illac C., Ledoux A., Fortin P.Y., de Barros S., Vabre C., Despas F., Peries S., Casaroli C., Bouvier C., Aubert S., de Pinieux G., Larousserie F., Galmiche L., Talmont F., Pitson S., Maddelein M.L, & Cuvillier O. (2022). Sphingosine Kinase-1 Is Overexpressed and Correlates with Hypoxia in Osteosarcoma: Relationship with Clinicopathological Parameters. Cancers, 14(3), 499.

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Comprehensive Antibody Panel for Hypoxia Signaling

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The primary antibodies used were: mouse anti-HIF1α (BD Biosciences), rabbit anti-HIF1α (D2U3T), rabbit anti-NDRG1 (D8G9), rabbit anti-BNIP3 (D7U1T), rabbit anti-ATF4 (D4B8), mouse anti-LAMP1 (D4O1S) (Cell-signalling technology), rabbit anti-ERK2 (C14), mouse anti-GAPDH (6C5), goat anti- LDHA (N14) (Santa Cruz Biotechnologies), mouse anti-MITF (C5) (Millipore), mouse- anti-MITF (D5) (Dako), rabbit anti-MITF (HPA003259), rabbit anti-SDHB (HPA002868) (Cambridge Biosciences). rabbit anti-HIF1α and anti-HIF2α used in ChIP-seq experiments were provided by the Ratcliffe lab, rabbit anti-HIF1β (Novus Biologicals), mouse anti-FLAG (M2) (Sigma) and mouse anti-HA (12CA5) (Roche). Alexa Fluor-conjugated secondary antibodies were obtained from Invitrogen.

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Western Blot Antibody Validation

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All reagents were from Sigma unless otherwise stated. Standard protocols were used as previously described57 (link)58 (link). Mouse anti-β-actin, 1:10000, Sigma, A5441; Mouse anti-HIF-1α,1:1000, BD Pharmingen, 610958; Rabbit anti-REST, 1:1000, Abcam, ab28018; Rabbit anti-Lamin A/C, 1:1000, Cell Signalling, 2032; Mouse anti-α-Tubulin, 1:2000, SCB, sc-8035.

Cavadas M.A., Mesnieres M., Crifo B., Manresa M.C., Selfridge A.C., Keogh C.E., Fabian Z., Scholz C.C., Nolan K.A., Rocha L.M., Tambuwala M.M., Brown S., Wdowicz A., Corbett D., Murphy K.J., Godson C., Cummins E.P., Taylor C.T, & Cheong A. (2016). REST is a hypoxia-responsive transcriptional repressor. Scientific Reports, 6, 31355.

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Immunoblot Analysis of HIF, Glycolysis, and mTOR Signaling

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Rabbit anti-HIF-2α (Novus, Littleton, CO, USA), mouse anti-HIF-1α (BD, San Jose, CA, USA), rabbit anti-GLUT-1 (ThermoScientific, Villebon-sur-Yvette, France), mouse anti-cyclin D1, rabbit anti-p70S6K, rabbit anti-phospho-p70S6K (Thr389), rabbit anti-phospho-p70S6K (Thr421/Ser424), rabbit anti-mTOR, rabbit anti-phospho-mTOR (Ser2448), rabbit anti-4E-BP1, rabbit anti-phospho 4E-BP1 (Ser65), rabbit anti-Akt, rabbit anti-phospho-Akt (Ser473) (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-Spns2 (Sigma), anti-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-puromycin (Millipore, 12D10) were used as primary antibodies. Proteins were visualized by an ECL detection system (Perbio, Villebon-sur-Yvette, France) using anti-rabbit or anti-mouse horseradish peroxidase-conjugated IgG (Bio-Rad, Marnes-la-Coquette, France). Densitometry quantitation was determined using the Image J software (NIH, Bethesda, MD, USA).

Bouquerel P., Gstalder C., Müller D., Laurent J., Brizuela L., Sabbadini R.A., Malavaud B., Pyronnet S., Martineau Y., Ader I, & Cuvillier O. (2016). Essential role for SphK1/S1P signaling to regulate hypoxia-inducible factor 2α expression and activity in cancer. Oncogenesis, 5(3), e209-.

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Hypoxia-Induced HIF-1α Expression

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For the Western blot analysis of hypoxia inducible factor-1α (HIF-1α), cells were incubated in 21% or 1% O2 for 72 h. Briefly, samples were collected in RIPA (RadioImmunoPrecipitation Assay) lysis buffer (50 mM Tris Hydrocloride, pH 7.9, 120 mM NaCl, 1% Nonidet, 1 mM EDTA, 5 mM NaF, 1 mM sodium othovanadate, 0.04 mM 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride), and protein concentration was determined by BCA Assay (BicinChoninic Acid)(Bio-Rad, München, Germany). Western blot analysis was performed using the following antibodies: mouse anti-HIF-1α diluted ¹/₅₀₀ (BD Bioscience, San Jose, CA, USA) and rabbit anti-actin diluted ¹/₅₀₀₀ (Santa Cruz, Dallas, TX, USA).

Bailleul Q., Navarin P., Arcicasa M., Bal-Mahieu C., Carcaboso A.M., Le Bourhis X., Furlan A., Meignan S, & Leblond P. (2021). Evofosfamide Is Effective against Pediatric Aggressive Glioma Cell Lines in Hypoxic Conditions and Potentiates the Effect of Cytotoxic Chemotherapy and Ionizing Radiations. Cancers, 13(8), 1804.

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