PA1-16601

antibody from Invitrogen Antibodies

Targeting: HIF1A bHLHe78, HIF-1alpha, HIF1, MOP1, PASD8

Provider product page for PA1-16601

Western blot

Immunocytochemistry

Immunoprecipitation

Immunohistochemistry

Flow cytometry

Chromatin Immunoprecipitation

Other assay

Antibody data

Antibody Data
Antigen structure
References [31]
Comments [0]
Validations
Western blot [5]
Immunocytochemistry [2]
Immunohistochemistry [2]
Other assay [21]

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Product number: PA1-16601 - Provider product page
Provider: Invitrogen Antibodies
Product name: HIF1A Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Other
Description: This antibody detects upregulation of HIF-1 alpha in hypoxic samples. Recommended positive controls: Human pancreas whole tissue lysate, COS-7 nuclear hypoxic induced cell lysate, human bone marrow whole tissue lysate (adult whole diabetes), HeLa hypoxic (CoCl2) cell lysate, HepG2 hypoxic (CoCl2) cell lysate, HeLa hypoxic cell Lysate, HepG2 hypoxic cell lysate, HIF-1 alpha knockout HeLa cell lysate
Reactivity: Human, Mouse, Rat, Canine, Goat, Hamster, Porcine, Rabbit
Host: Rabbit
Isotype: IgG
Vial size: 100 µL
Concentration: 1 mg/mL
Storage: -20°C

HIF1A protein structure - PA1-16601 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of HIF-1 alpha was performed by loading 50 µg of Cos7 cell lysates under hypoxic (Lane 1: CoCl2 treated) or untreated (Lane 2) conditions onto a gel and then transferring blotted to membrane. Blot was probed with a rabbit polyclonal antibody recognizing HIF-1 alpha (Product # PA1-16601) and chemiluminescent deterction performed using ECL.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of HIF1A in 50 µg Cos-7 nuclear extract. Samples were incubated in HIF1A polyclonal antibody (Product # PA1-16601). Lane 1: CoCl2 treated Cos-7 nuclear extract; (hypoxic); Lane 2: Untreated Cos-7 nuclear extract (normoxic). 10 second ECL exposure.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of HIF1A in A. PC12 (rat) cells, 20% oxygen and B. PC12 (rat) cells, 1% oxygen. Samples were incubated in HIF1A polyclonal antibody (Product # PA1-16601).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of HIF1A in 50 µg Cos-7 nuclear extract. Samples were incubated in HIF1A polyclonal antibody (Product # PA1-16601). Lane 1: CoCl2 treated Cos-7 nuclear extract; (hypoxic); Lane 2: Untreated Cos-7 nuclear extract (normoxic). 10 second ECL exposure.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of HIF1A in 0.5 mg/mL lysates of MCF +/- DMOG, BioSpherix MSCs in hypoxic conditions, and BioSpherix MSCs in normoxic conditions. Samples were incubated in HIF1A polyclonal antibody (Product # PA1-16601) using a dilution of 1:100 followed by a HRP-conjugated Anti-Rabbit secondary antibody. A band was detected for HIF-1 alpha at approximately 116 kDa (as indicated). This experiment was conducted under standard assay conditions, and using the 12-230 kDa separation module. Non-specific interaction with the 230 kDa Simple Western standard seen with this antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Submitted by: Invitrogen Antibodies (provider)
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Submitted by: Invitrogen Antibodies (provider)
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Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1 Effects of Rg5 on adipose hypoxia. (A) Adipose hypoxia examination with pimonidazole staining in HFD mice. (B) HIF-1alpha protein expression in adipose tissue of HFD-fed mice. (C,D) HIF-1alpha protein expression in differentiated adipocytes treated with palmitate (PA), 1% O 2 for 8 h. (E,F) Pimonidazole staining in differentiated adipocytes treated with PA for 8 h or 1% O 2 for 4 h. The results were expressed as the mean +- SD of three independent experiments. * p < 0.05 vs . Control (Ctr), # p < 0.05 vs . Blank. TU, Tauroursodeoxycholic Acid; Met, Metformin.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 8 HIF-1alpha protein levels were stabilized by AA treatment. HIF-1alpha protein levels were evaluated by western blot at different time-points upon AA treatment. (A) mESC incubated for 14h with CoCl 2 (300 uM) a known disruptor of prolyl hydroxylase activity, show increased Hif-1alpha protein levels. mESC without CoCl 2 treatment were used as control and different protein volumes were loaded in the gel. (B) Representative blot for E14 cells showing increased Hif-1alpha protein levels downstream to AA treatment. (C) Densiometric evaluation of HIF-1alpha and actin (loading control) bands show that a statistical significant difference exists between control and treated conditions at 2 and 4 hours for both E14 and R1 mESC. Data shown as protein level fold increase for each one of the AA conditions relative to the respective control. Data were normal and evaluated by one sample T Student tests against a theoretical value of 1. Error bars = SEM and statistical significance is considered when * p

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 Accumulation of HIF-1alpha in cultured CjECs results in the secretion of angiogenic mediators. (A) Immunoblot for HIF-1alpha in ihCjECs exposed to hypoxia (1% O 2 ) or a HIF inducer (100 muM DFO or 300 muM DMOG), in normoxia (20% O 2 ) for 4 hours. One hundred nanomolar digoxin was used to inhibit HIF-1alpha accumulation. (B) Immunoblot for HIF-1alpha in prCjECs exposed to 1% O 2 or 300 muM DMOG for 4 hours. (C) H&E stain of stratified prCjECs grown on vitrigel. (D) Immunoblot for HIF-1alpha in stratified prCjECs grown on vitrigel exposed to 1% O 2 or 300 muM DMOG for 4 hours. (E-H) EC tubule formation by HMVECs treated with conditioned media from ihCjECs exposed to 1% O 2 (E, F) or 100 muM DFO or 300 muM DMOG (G, H), in the absence (E, G) or presence (F, H) of 100 nM digoxin, compared to media conditioned by cells exposed to 20% O 2 for 24 hours. Ten percent FBS was used as a positive control.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig 10 Immunofluorescence staining (Alexa Fluor 594) for Ang-1 and HIF-1alpha, 15 days after implantation. Scale bar 100 mum. ( A ) SiONPx2. ( B ) Uncoated ""bare"" implant. ( C ) Percentage of area occupied by Ang-1. Statistical difference, *** p < 0.001. ( D ) SiONPx2. ( E ) Uncoated ""bare"" implant. ( F ) Percentage of area occupied by HIF-1alpha. Statistical difference, * p < 0.05. Ang-1 = Angiopoietin-1; HIF-1alpha = hypoxia inducible factor-1 alpha; SiONP = silicon oxynitrophosphide.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5 LW1497 suppressed lung cancer progression in a lung cancer orthotopic mouse model. ( A ) In vivo experimental protocol of A549 cells in NOD/SCID mouse using LW1497 drug as described in Methods section. ( B ) Hematoxylin and eosin staining of the lungs in LW1497- or vehicle-treated NOD/SCID mice injected with A549 cells. Lungs were analyzed by hematoxylin and eosin staining. ( C ) Comparison of tumor areas: mice treated with LW1497 versus mice treated with vehicle. Tumor areas were measured by intravital microscopic analysis (Axiotech Vario microscope, Zeiss, Germany). * p < 0.05, ** p < 0.01 compared with the control group. ( D ) Immunohistochemical analysis of Ki67, HIF-1a, Slug, and EMT markers including Vimentin (Vim), E-cadherin (E-cad), and N-cadherin (N-cad) in lung tumors in LW1497 (10, 30 mg/kg)- or vehicle-treated mice orthotopically injected with A549 cells into the lung.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2 Human NPCs have the function of recovering damaged R28 cells. R28 cells were cultured with NPCs or hPSCs 3 h prior to CoCl 2 treatment. Then, the R28 cells were treated with CoCl 2 (300 muM). ( A ) Viability assays performed after 24 h. Data are expressed as percentage (mean +- SEM) of viable cells compared to those in the control group. Significantly different values between groups are indicated by different letters ( p < 0.05). ( B ) Apoptosis-related protein expressions were also determined. ( C ) Western blot analyses of target protein expression levels, using R28 lysates with CoCl 2 . The quantified values of target protein expression are presented (bottom panel) (* p < 0.05 vs. the control; # p < 0.05, ## p < 0.01 vs. CoCl 2; + p < 0.05 vs. PSCs).

Supportive validation

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Experimental details: FIG. 14. PI3K/HIF-1alpha/VEGF pathway in macrophages exposed to ferryl hemoglobin. (A) Human macrophages were treated with 10 mu M oxyHb or ferrylHb for 2 h. The expression of PI3K and phopsho-PI3K protein was assessed by Western blot ( n = 3). (B) Human macrophages were treated with 10 mu M heme, oxyHb, metHb, or ferrylHb for 16 h. The expression of HIF-1alpha was assessed by Western blot ( n = 5). (C) Human macrophages were treated with 10 mu M oxyHb or ferrylHb for 24 h. The secreted VEGF-A was measured by the ELISA-kit. * p < 0.5; ** p < 0.01. ELISA, enzyme-linked immunosorbent assay; HIF-1alpha, hypoxia-inducible factor 1-alpha; PI3K, phosphoinositide 3-kinase; VEGF, vascular endothelial growth factor.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: CoCl 2 -induced hypoxia differentially affects HIF-1alpha expression in cementoblasts. ( A , B ) RT-qPCR quantification of HIF-1alpha gene expression in OCCM-30 cells cultured under normoxia or hypoxia (100, 400 muM) conditions for indicated timepoints. The relative mRNA expression of HIF-1alpha was obtained through normalizing to internal PPIB. Each dot, triangle and square symbol represent a relative PD-L1 expression value. ( C , D ) HIF-1alpha protein levels were determined by Western blot. Internal beta-actin levels served as loading control. Quantification indicated the enhanced HIF-1alpha expression in 8 to 24 h in the 400 muM hypoxia treated group relative to the control group. Results are reported as fold change and relative the control group as 1. ( E ) Confocal microscopy analysis of subcellular localization of HIF-1alpha (red) in OCCM-30 cells cultured under normoxia or hypoxic conditions for 24 h at the same initial cell density. White arrows show its cellular localization. Nuclei are stained with DAPI (blue). Individual and merged images of HIF-1alpha and DAPI are shown. Scale bar: 100 mum. The blots and photomicrographs represent three independent experiments with SD. Statistically significant differences (indicated by asterisks) are indicated as follows (ns, no significant difference; * p < 0.05; ** p < 0.005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 A major role of HIF-1alpha inhibition on HIF-1alpha expression under hypoxia. Cells were preincubated with different concentrations of HIF-1alpha inhibitor (IDF11774) for 2 h and then incubated in hypoxic conditions for 24 h, respectively. ( A ) Western blot analysis of the expression levels of HIF-1alpha in the presence or absence of IDF-11774 (0, 5, 10, 20 nM). HIF-1alpha accumulation was reduced by the increased concentration of IDF-11774. Internal beta-actin levels serve as loading control. ( B ) Quantification was performed to detected protein changes of HIF-1alpha, and it was demonstrated that its expression is significantly inhibited by the highest concentration of IDF-11774 (20 nM). ( C , D ) Representative IF staining showing the distribution changes of HIF-1alpha in hypoxia-treated OCCM-30 cells with the treatment of the pharmacological signaling inhibitor IDF-11774 (20 nM). Yellow arrows indicate the expression of HIF-1alpha was effectively inhibited by the HIF-1alpha inhibitor IDF-11774. ( E ) Verification of the efficacy of HIF-1alpha CRISPR/Cas9 system. Knockdown of HIF-1alpha gene expression by CRISPR/Cas9 decreased the hypoxia-induced gene expression of HIF-1alpha in OCCM-30 cells. The blots and photomicrographs represent three independent experiments with SD. Statistically significant differences (indicated by asterisks) are indicated as follows (ns, no significant difference; * p < 0.05; ** p < 0.005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5. Yap1 and Taz inhibit HIF-1alpha-controlled gene expression. ( A ) Increased expression of the HIF1alpha target genes VEGFA and ANGPTL4 as well as Yap1/Taz target genes CTGF and CYR61 under hypoxic condition (1% O 2 ) compared to normoxia (21% O 2 ) (n = 4; data are presented as mean +-sem, P values, two-tailed unpaired t-test ). ( B ) Confocal image of HUVEC showing nuclear accumulation of Yap1/Taz in 1% O 2 relative to 21% O 2 . Nuclei, DAPI (blue). ( C ) Increased expression of the Yap1/Taz target genes CTGF, CYR61 and the HIF1alpha target VEGFA in HUVECs in 1% O 2 relative to 21% O 2 . CTGF and CYR61 expression under both conditions is significantly reduced in siYAP1/TAZ -transfected (Y/T) HUVECs. VEGFA expression significantly increased in Y/T cells in both conditions (n = 4; data are presented as mean +-sem, P values, two-tailed unpaired t-test ). ( D ) Western blots showing immunoprecipitation (IP) of Yap1/Taz followed by immunoblotting of HIF1alpha and Yap1/Taz. IgG is used as negative control in IP. Lysates are shown as loading control. ( E ) Increased VEGFA , ANGPTL4 , IGFBP2 expression in siYAP1/TAZ -treated HUVECs is normalized by siHIF1A transfection, whereas baseline VEGFA , ANGPTL4 and IGFBP2 is not altered by HIF1A knockdown alone (n = 3-4; data are presented as mean +-sem, P values, two-tailed unpaired t-test ). ( F ) Enrichment of VEGFA promoter sequences after chromatin immunoprecipitation with HIF1alpha antibodies compared to IgG antibodies under

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6 Chloramphenicol interrupted protein interaction between SENP-1 and HIF-1alpha and promoted HIF-1alpha degradation via the autophagy pathway. ( A ) Representative images of co-immunoprecipitation assays performed with HIF-1alpha-overexpressing H1299 cells in the presence or absence of chloramphenicol. Whole cell lysates was immunoprecipitated with anti-HIF-1alpha (left) or anti-SENP-1 (right) antibody, followed by immunoblotting analysis. A protein complex of HIF-1alpha/SENP-1 was found in the control treatment. Chloramphenicol treatment not only reduced HIF-1alpha levels in whole lysate, but also inhibited HIF-1alpha/SENP-1 interaction. ( B ) Whole lysate of HIF-1alpha-overexpressing H1299 was prepared and aliquoted (1 mg protein/vial). Before reaction with the captured antibody, one vial of sample was incubated with chloramphenicol (100 mug/mL) at 4 degC for 1 h. Another vial, without chloramphenicol treatment, was used as a control. Then, the co-immunoprecipitation was performed as described previously. The input data showed that neither HIF-1 alpha nor SENP-1 protein level was changed in whole lysates, whatever the presence or absence of chloramphenicol treatment. The HIF-1alpha/SENP-1 protein complex, which was clearly found in lysate without the chloramphenicol treatment, was diminished in lysate with the chloramphenicol treatment. This data provides substantial evidence to support the interaction between HIF-1alpha and SENP-1 was damaged by chloramphenicol. (