- 700505 - Invitrogen Antibodies HIF1A antibody

Submitted references Human induced pluripotent stem cell-based platform for modeling cardiac ischemia.

Häkli M, Kreutzer J, Mäki AJ, Välimäki H, Lappi H, Huhtala H, Kallio P, Aalto-Setälä K, Pekkanen-Mattila M
Scientific reports 2021 Feb 18;11(1):4153

CoCl(2) simulated hypoxia induce cell proliferation and alter the expression pattern of hypoxia associated genes involved in angiogenesis and apoptosis.

Rana NK, Singh P, Koch B
Biological research 2019 Mar 15;52(1):12

Reference Genes for Addressing Gene Expression of Bladder Cancer Cell Models under Hypoxia: A Step Towards Transcriptomic Studies.

Lima L, Gaiteiro C, Peixoto A, Soares J, Neves M, Santos LL, Ferreira JA
PloS one 2016;11(11):e0166120

Thyroid Transcription Factor 1 Reprograms Angiogenic Activities of Secretome.

Wood LW, Cox NI, Phelps CA, Lai SC, Poddar A, Talbot C Jr, Mu D
Scientific reports 2016 Feb 25;6:19857

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of HIF1-alpha was performed by loading 30 µg of HeLa, U-2 OS, Jurkat, U-87 MG, A549, A431, HEK-293, and MDA-MB-231 cell lysates using Novex®NuPAGE®4-12% Bis-Tris gel (Product # NP0321BOX), Xcell SureLock Electrophoresis system (Product # EI0002), Novex sharp Pre-stained Protein Standard (Product # LC5800). Proteins were transferred to a PVDF membrane and blocked with 5% skim milk for 1 hour at room temperature. HIF1-alpha was detected at ~93 kDa using HIF1-alpha Recombinant Rabbit Monoclonal Antibody (Product # 700505) at a 1:1000 dilution in 2.5% skim milk at 4° C overnight on a rocking platform. Detection was performed using an HRP-conjugated Goat anti-Rabbit secondary antibody (Product # G-21234) at a 1:5000 dilution and chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of HIF1-alpha was performed on 70% confluent log phase HeLa cells. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0. 25% Triton X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature. The cells were labeled with HIF1-alpha Recombinant Rabbit Monoclonal Antibody (Product # 700505) at a dilution of 1:400 in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Fluor® 488 Goat anti-Rabbit IgG secondary antibody (Product # A-11008) at a dilution of 1:400 for 30 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938) F-actin (Panel c: red) was stained with Alexa Fluor® 594 phalloidin (Product # A12381) and panel d is a merged image showing cytoplasmic localization. Panel e is a control without primary antibody. The images were captured using a Nikon microscope at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Flow cytometry analysis of HIF1-alpha was performed on HeLa cells. Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0. 25% Tritonª X-100 for 20 minutes, and blocked with 5% BSA for 1 hour at room temperature. Cells were labeled with ABfinityª HIF1-alpha recombinant rabbit monoclonal antibody (Product # 700505, red histogram) or with rabbit isotype control (pink histogram) at a dilution of 1:400 in 2.5% BSA. After incubation at room temperature for 3 hours, the cells were labeled with Alexa Fluor¨ 488 goat anti-Rabbit Secondary antibody (Product # A11008) at a dilution of 1:400 for 30 minutes at room temperature. The representative 10,000 cells were acquired and analyzed for each sample using an Attune¨ Acoustic Focusing Cytometer. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Chromatin immunoprecipitation analysis of HIF-1 alpha was performed using cross-linked chromatin from 1 x 10^6 HCT116 human colon carcinoma cells treated with serum for 0, 15, and 30 minutes. Immunoprecipitation was performed using a multiplex microplate Matrix ChIP assay (see reference for Matrix ChIP protocol: http://www.ncbi.nlm.nih.gov/pubmed/22098709) with 1.0 µL/100 µL well volume of a HIF-1 alpha rabbit monoclonal antibody (Product # 700505). Chromatin aliquots from ~1 x 10^5 cells were used per ChIP pull-down. Quantitative PCR data were done in quadruplicate using 1 µL of eluted DNA in 2 µL SYBR real-time PCR reactions containing primers to amplify -15kb upstream of the human Egr-1 locus, or exon-1 or exon-2 of Egr-1. PCR calibration curves were generated for each primer pair from a dilution series of sheared total genomic DNA. Quantitation of immunoprecipitated chromatin is presented as signal relative to the total amount of input chromatin. Results represent the mean +/- SEM for three experiments. A schematic representation of the Egr-1 locus is shown above the data where boxes represent exons (black boxes = translated regions, white boxes = untranslated regions), the zigzag line represents an intron, and the straight line represents upstream sequence. Regions amplified by Egr-1 primers are represented by black bars. Data courtesy of the Innovators Program.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6 The VEGF upregulated by TTF-1 is present in exosomes in a hypoxia-independent manner. Western blots of NCI-H1299 cells subject to hypoxia or DFO show an increase of HIF1alpha ( A ). VEGF was quantified in the CM of NCI-H1299 (TTF-1 - ) and NCI-H2009 (TTF-1 + ) under normoxia, hypoxia, or DFO treatment ( B ). The CM of the A549 cells retrovirally transfected with EV , HDD , or TTF-1 was analyzed by VEGF ELISA assays after three types of treatments, normoxia, hypoxia, or DFO ( C ). The exosomes and EDMs of A549-based transfectant cells were analyzed for CD9 protein expression by immunoblotting ( D ) and VEGF ELISA assays ( E ).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 4 ( a ) HIF1alpha expression in control (n = 26) samples and in 6 h (n = 4), 8 h (n = 6), 10 h (n = 3), 12 h (n = 3) and 24 h (n = 6) hypoxia or 8 h (n = 4) and 24 h (n = 4) hypoxia-reoxygenation time points from western blot analysis presented as mean + standard deviation. During the first 12 h of hypoxia, there seems to be no significant changes in the expression of the protein, although in 6 h and 12 h samples there is a slight increase. On the other hand, after 24 h of hypoxia, as well as after 8 h hypoxia-reoxygenation there is a statistically significant decrease in the HIF1alpha expression. ( b ) Expression of MyBPC3 and Troponin T decreases during 8 h (n = 6) and 24 h (n = 6) hypoxia, and 8 h (n = 4) and 24 h (n = 4) hypoxia-reoxygenation compared to control (n = 20) samples. The expression is especially low in 24 h hypoxia-reoxygenation samples but has decreased also in other hypoxia samples. ( c ) Examples of the western blot bands. *p < 0.05; **p < 0.01; ***p < 0.001.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 5 Western blot analysis of beta -actin, HIF-1alpha, VEGF, p53 and BAX protein in breast cancer cells grown under hypoxia (treated with CoCl 2 ) as well as in normoxia (without treatment). a Differential expression pattern of HIF-1alpha and VEGF protein isolated from MCF-7 cells. b Shows the dose dependent enhancement in expression of hypoxia associated proteins HIF-1alpha, VEGF, p53 and BAX in MDA-MB-231 cells grown under hypoxic condition, P < 0.05

700505

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