Luseogliflozin was provided by Taisho Pharma, Co. (Tokyo, Japan). An anti-HIF-1α antibody was obtained from Novus Biologicals, Inc. (Littleton, CO, USA). Anti-AMP-activated protein kinase (AMPK)-α and anti-phosphorylated (p)-AMPKα (Thr 172) antibodies were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA). An anti-nucleoporin p62 antibody was obtained from BD Bioscience Japan, Inc. (Tokyo, Japan), and an anti-fibronectin antibody was obtained from Merck, Inc. (Kenilworth, NJ, USA). Alexa Fluor 594 donkey anti-mouse and Alexa Fluor 488 donkey anti-rabbit secondary antibodies were purchased from Invitrogen (Carlsbad, CA, USA). AICAR was purchased from Calbiochem (San Diego, CA, USA), and other chemicals and antibodies were obtained from Merck, Inc.
Anti hif 1α antibody
Manufactured by Novus Biologicals
Sourced in United States, United Kingdom
The Anti-HIF-1α antibody is a tool used in research to detect and quantify the presence of the HIF-1α protein in biological samples. HIF-1α is a transcription factor that plays a crucial role in the cellular response to hypoxic conditions.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin antibody, by Merck Group (3 mentions)
Anti β actin antibody, by Santa Cruz Biotechnology (2 mentions)
Imprint chromatin immunoprecipitation kit, by Merck Group (2 mentions)
Ripa lysis buffer, by Cell Signaling Technology (2 mentions)
Ani tubulin antibody, by Cell Signaling Technology (2 mentions)
Protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Anti phosphorylated ampkα thr 172, by Cell Signaling Technology (1 mentions)
Alexa fluor 488 donkey anti rabbit, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 donkey anti mouse, by Thermo Fisher Scientific (1 mentions)
Ecl plus reagent, by PerkinElmer (1 mentions)
Imagequant las 4000 imager, by GE Healthcare (1 mentions)
Polyvinylidene difluoride pvdf membrane, by Bio-Rad (1 mentions)
Las 4000, by Cytiva (1 mentions)
Anti sirt1, by Cell Signaling Technology (1 mentions)
Anti acetyl lysine, by Cell Signaling Technology (1 mentions)
Protein a g plus agarose, by Santa Cruz Biotechnology (1 mentions)
Anti sirt1 antibody, by Cell Signaling Technology (1 mentions)
Anti hif 1α, by Novus Biologicals (1 mentions)
Em 10a, by Zeiss (1 mentions)
Anti pp2ac, by Merck Group (1 mentions)
31 protocols using anti hif 1α antibody
1
Luseogliflozin Regulates HIF-1α and AMPK
2
HIF-1α Expression in PC12 Cells
3
Sirt1 and HIF-1α Immunoprecipitation
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Approximately 1 mg of total proteins was incubated overnight at 4°C with anti‐Sirt1 antibody (Cell Signaling) or anti‐HIF‐1α antibody (Novus Biologicals, Littleton, CO, USA) followed by precipitation with 20 µl of protein A/G‐Plus‐Agarose (Santa Cruz, Dallas, TX, USA) for 4 hr at 4°C. The precipitated complexes were immunoblotted with anti‐Sirt1 (Cell Signaling), anti‐HIF‐1α (Novus Biologicals), or anti‐acetyl‐lysine (Cell Signaling).
4
Intracellular Localization of HIF-1α
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To evaluate in more detail the intracellular localization of HIF-1α within DH82Ond pi cells, immunoelectron microscopy was performed using a 10% neutral buffered formalin fixed cell pellet as previously described [50 (link)]. Ultrathin sections of LR-White embedded samples were immunolabeled with an anti-HIF-1α antibody (1:500 dilution; Novus biologicals) followed by a goat anti-rabbit IgG 10 nm immunogold conjugated secondary antibody (BBI Solutions, Crumlin, United Kingdom). Samples were further evaluated using a transmission electron microscope (EM 10A, Carl Zeiss Microscopy GmbH, Jena, Germany) equipped with a 2K-CCD-Camera (TRS) and using Image SP professional software.
5
Regulation of Endothelial Nitric Oxide Synthase
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Fura-2 AM was purchased from Teflabs (Austin, TX, USA) and Cal-520 AM from Stratech (Newmarket, United Kingdom). eNOS, PP2A-A (α/β), and PR72/130 antibodies were from Santa Cruz Biotechnologies (Dallas, TX, USA). Anti-PP2A-C and α-tubulin were from Millipore-Sigma (Watford, United Kingdom). Anti-phospho-eNOS S1177, T495, and S633; anti-phospho-AMPK T172; and total α-AMPK, anti-phospho-protein kinase B (Akt) S473 and total Akt, anti-phospho-ERK1/2 (T204/202) and total ERK1/2 antibodies were from Cell Signaling Technology (Danvers, MA, USA). Anti-HIF1-α antibody was from Novus Biologicals (Littleton, CO, USA). cGMP ELISA was from Cayman Chemical (Ann Arbor, MI, USA). The anti-SERCA-2 antibody (2D8) was kindly provided by Dr. Kalwant Authi (Cardiovascular Division, King’s College London). On-Target Plus PP2A-C (human) and control scrambled siRNA were from Dharmacon (Lafayette, CO, USA). All other chemicals were purchased from Millipore-Sigma.
6
Western Blot Analysis of HIF-1α
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Samples, separated by a 10% SDS-PAGE, were then transferred to the polyvinylidene difluoride membrane (GE healthcare, Pittsburgh, PA, USA). The membrane was incubated with the blocking solution, containing milk, and then further washed using 0.1% and 0.3% TTBS, containing Tween-20 either 0.1% or 0.3% in Tris-buffered saline, sequentially. The anti-HIF-1α antibody (Novus Biologicals, Littleton, CO, USA) was purchased. The enhanced ECL detection system (GE healthcare) was used. Signals were detected using a Storm phosphoimager (Molecular Dynamics, Caesarea, Israel).
7
Cell Fractionation and Western Blot Analysis
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For cell lysis, RIPA buffer (50mM Tris-HCl at pH 8.0; 0.1% SDS; 0.5% sodium deoxycholate; 150mM NaCl; 1% NP-40; protease inhibitors) and whole cell lysate buffer (0.1mM EDTA; 400mM NaCl; 10mM HEPES at pH 7.9; 0.1mM EDTA; 1mM DTT; 5% glycerol; protease inhibitors) were used. Nuclear and cytoplasmic fractions were extracted with the NE-PER Nuclear and Cytoplasmic Extraction Reagents kit (Thermo Scientific, Rockford, IL, USA). Anti-HIF-1α antibody (1:1000) and anti-integrin αVβ6 antibody (1:1000) were obtained from Novus Biologicals (Littleton, CO, USA). Antibodies to E-cadherin (1:5000), phospho-p65 (1:1000), p65 (1:1000), phospho-ERK1/2 (1:1000), ERK1/2 (1:1000), phospho-p38 (1:1000), p38 (1:1000), phospho-JNK (1:1000), JNK (1:1000), phospho-STAT3 (1:1000), STAT3 (1:1000), histone H3 (1:1000), and GAPDH (1:1000) were purchased from Cell Signaling (Beverly, MA, USA). Anti-β-actin antibody (1:5000) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The samples were normalized to β-actin using Image J software.
8
USP8-mediated Nrdp1 regulation in HIF-1α signaling
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After the indicated treatments, PC12 cells were lysed in RIPA buffer and centrifuged at 12,000× g for 10 min at 4°C. The supernatant were incubated overnight at 4°C with 4 μg of anti-USP8 (Santa Cruz Biotech, Chicago, IL, USA), followed by precipitation with 50 μl of Dynabeads protein A (Pierce Biotechnology, Rockford, IL, USA) for 10 min at room temperature. The protein A were then washed extensively with binding buffer, resuspended in SDS-PAGE buffer, and boiled for 5 min. Samples of 30 μg total cell lysate were used as an input control. The precipitated complexes were separated on SDS-PAGE gels, and transferred to nitrocellulose membranes, and immunoblotted with anti-Nrdp1 (BETHYL Laboratories, Montgomery, TX, USA), K48-linkage-specific anti-ubiquitin antibody (Abcam, Cambridge, MA, USA) or anti-HIF-1α antibody (Novus Biological, Littleton, CO, USA) to detect the presence of these proteins in the complex. Normal rabbit IgG (Santa Cruz Biotech) was used as a loading control.
9
UVA-Induced HIF Regulation in hAMSCs
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hAMSCs were irradiated with the indicated doses of UVA and then incubated with the indicated concentrations of aspartic acid for one hour or three days under serum-free conditions. The cells were washed twice with cold PBS (Sigma—Aldrich, St. Louis, MO, USA) and then lysed in 150 μl of sample buffer (100 mM Tris-HCl), pH 6.8, 10% glycerol, 4% sodium dodecyl sulfate (SDS), 1% bromophenol blue, 10% β-mercaptoethanol (All reagents were from Sigma—Aldrich, St. Louis, MO, USA). Next, the samples were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Immobilon-P PVDF membranes (Millipore Corporation, Bedford, MA, USA). The membranes were incubated overnight at 4°C with anti-HIF-1α antibody (Novus Biologicals, Beverly, MA, USA), anti-HIF-2α antibody (Novus Biologicals, Beverly, MA, USA), and anti-β-actin antibody (Sigma—Aldrich, St. Louis, MO, USA). The membranes were subsequently washed three times with Tris-buffered saline containing Tween-20 (TBST) (Sigma—Aldrich, St. Louis, MO, USA) probed with horseradish peroxidase-conjugated secondary antibody (Sigma—Aldrich, St. Louis, MO, USA), and developed using an ECL (enhanced chemiluminescence) Western blotting detection system (Amersham Biosciences).
10
Immunohistochemical Analysis of HIF-1α
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After dewaxing and hydration, the slides were incubated in citrate antigen-repairing solution and placed in a microwave oven at high power for 10 min. The solution was then allowed to cool at room temperature for 15 min, followed by washing in PBS for 5 min. To block the activity of endogenous peroxidase, the slides were incubated in 3% hydrogen peroxide in methanol for 10 min at room temperature. To block the nonspecific binding, the slides were incubated with 5% normal goat serum in PBS for 30 min. After the step listed above, the slices were incubated with an anti-HIF-1α antibody (1:50, Novus Biologicals, USA) in PBS and then incubated with an HRP-conjugated secondary goat-anti mouse antibody (1:100, Abcam, UK) in PBS for 1 h. The slices were developed with DAB and counterstained with haematoxylin. The immunoreactive specificity was confirmed by omitting the primary antibody. The pyramidal cells in the CA1 region were examined.
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