Clicking the images or links will redirect you to a website hosted by BenchSci that provides third-party scientific content. Neither the content nor the BenchSci technology and processes for selection have been evaluated by us; we are providing them as-is and without warranty of any kind, including for use or application of the Thermo Fisher Scientific products presented. This Antibody was verified by Knockout to ensure that the antibody binds to the antigen stated.查看细节    Knockout of HIF1A was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, Assay ID CRISPR970881_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Western blot analysis of HIF1A was performed by loading 30 µg of HeLa wild type (Lane 1), HeLa wild type treated with 150uM CoCl2 for 48 hrs (Lane 2), HeLa Cas9 (Lane 3), HeLa Cas9 treated with 150uM CoCl2 for 48 hrs (Lane 4), HeLa HIF1A KO (Lane 5) and HeLa HIF1A KO treated with 150uM CoCl2 for 48 hrs (Lane 6) modified whole cell extracts. The samples were electrophoresed using NuPAGE™ Novex™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with Anti-HIF1A Monoclonal Antibody (mgc3) (Product # MA1-516, 1:2000 dilution) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using SuperSignal™ West Dura Extended Duration Substrate (Product # 34076). Loss of signal upon CRISPR mediated knockout (KO) using the LentiArray™ CRISPR product line confirms that antibody is specific to HIF1A. Uncharacterized bands were observed in HeLa Cas9 samples at ~40 kDa and 45 kDa.   Immunohistochemistry was performed on normal biopsies of deparaffinized Human tonsil tissue. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:20 with a mouse monoclonal antibody recognizing HIF-1 alpha (Product # MA1-516) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.   Immunofluorescent analysis of HIF-1 alpha (green) in A549 cells either left untreated (left panel) or treated with 100uM desferrioxamine (DFX) for ~16 hours (right panel). The cells were fixed with 4% Paraformaldehyde for 15 min at room temp, permeabilized with 0.1% Triton X-100 in TBS for 15 minutes at room temperature and blocked with 0.3% BSA for 15 minutes at room temperature. Cells were probed with a HIF-1 alpha monoclonal antibody (Product # MA1-516) at a dilution of 1:100 overnight at 4C, washed with PBS, and incubated with an Alexa Fluor 488-conjugated goat anti-mouse IgG secondary antibody (Product # R37120) at a dilution of 1:500 for 2 hours at room temperature. F-actin (red) was stained with Alexa Fluor 647 Phalloidin (Product # A22287) and nuclei (blue) were stained with DAPI (Product # P-36931). Images were taken on a Zeiss LSM 710 confocal microscope.   Immunofluorescent analysis of HIF-1 alpha (green) in HeLa cells either left untreated (left panel) or treated with 100uM Deferoxamine mesylate for ~16 hours (right panel). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 15 minutes at room temperature and blocked with 0.3% BSA for 15 minutes at room temperature. Cells were probed with a HIF-1 alpha monoclonal antibody (Product # MA1-516) at a dilution of 1:100 for at least 1 hour at room temperature, washed with PBS, and incubated with a DyLight 488-conjugated goat anti-mouse IgG secondary antibody (Product # 35502) at a dilution of 1:500 for 30 minutes at room temperature. F-actin (red) was stained with DyLight 594 Phalloidin (Product # 21836) and nuclei (blue) were stained with Hoechst 33342 dye (Product # 62249). Images were taken on a Thermo Scientific ArrayScan Instrument at 20X magnification.   Antibody specificity was demonstrated by CRISPR-Cas9 mediated knockout of target protein. A loss of signal was observed for target protein in HIF1A KO cell line compared to control cell line using Anti-HIF1A Monoclonal Antibody (mgc3) (Product # MA1-516). {KO}   Hypoxia controls Gal-1 expression in KS through HIF-independent, NF-κB–dependent mechanisms. (A–D) Expression of Gal-1 in KS cells transfected with or without HIF-1α siRNA or a super-repressor form of IκB-α (IκB-α-SR) and incubated under hypoxia or normoxia. (A) Promoter activity. (B) qRT-PCR of Gal-1 mRNA relative to RN18S1. AU, arbitrary units. Data are the mean ± SEM of five (A) or three (B) independent experiments. (C) Immunoblot of Gal-1, IκB-α, HIF-1α, and actin. Data are representative of four independent experiments. (D) ELISA of Gal-1 secretion. Data are the mean ± SEM of three independent experiments. (E) ELISA of Gal-1 secretion by KS cells cultured under hypoxic or normoxic conditions in the presence or absence of HIF-1α or NF-κB inhibitors. Data are the mean ± SEM of three independent experiments. (F and G) Immunoblot (F) and qRT-PCR (G) of Gal-1 expression induced by hypoxia (Hyp) in human and mouse melanoma (A375 and B16-F0), mouse breast carcinoma (4T1), and human prostate carcinoma (LNCaP) cell lines. Data are representative (F) or are the mean ± SEM (G) of three independent experiments. (H) Western blot of HIF-1α, IκB-α, Gal-1, and actin upon treatment of KS cells with CoCl2 (chemical activator of HIF-1α). Data are representative of four experiments. (I) Gal-1 promoter activity upon treatment of KS cells with CoCl2. Modulation of pGL3–Gal-1–Luciferase activity relative to Renilla expression is shown. Data are the mean ± SEM of three independent experiments. {KD}   Figure 1 Effect of Cadmium on HIF-1alpha accumulation and hypoxia preconditioning protection. A : Cells were incubated under normoxia with 200 muM CoCl 2 (as a positive control), 5 muM CdCl 2 alone, hypoxia (0.5% O 2 ) alone, or hypoxia with CdCl 2 for 4 h. Whole cell lysates were collected immediately after treatment, separated on SDS-PAGE gel and blotted for HIF-1alpha. beta-actin was detected as a loading control. B : Cells were pretreated with hypoxia with or without CdCl 2 for 4 h and irradiated 2 min with UV. Four hours after irradiation, cells were fixed and stained for TUNEL. Bar graph shows percentage of TUNEL positive cells in indicated groups. Error bars represent standard error of the mean (n=3); the asterisk indicates significantly different from UV control (p   The specificity of anti-HIF1-alpha antibody (Product # MA1-516) was demonstrated by the immunofluorescence detection of increased accumulation of HIF1-alpha in the nucleus in HeLa cells upon treatment with deferoxamine mesylate (right panel) compared to untreated cells (left panel). {TM}   The specificity of anti-HIF1-alpha antibody (Product # MA1-516) was demonstrated by the immunofluorescence detection of increased accumulation of HIF1-alpha in the nucleus in A549 cells upon treatment with deferoxamine mesylate (right panel) compared to untreated cells (left panel). {TM}   Figure 2 Effect of HIF-1α siRNA on HIF-1α protein expression. Cells were transfected with 15 and 25 nM HIF-1α siRNA and an siRNA non targeting control as indicated for 6 h. Twenty-four hours after transfection, cells were exposed to 4 h of hypoxia. Protein was collected immediately after treatment and blotted for HIF-1α. Bar graph represents the band intensity relative to the control. Error bars represent the standard error of the mean (n=3). β-actin is detected as loading control. {KD}   Figure 6 The expression levels of HIF-1alpha were detected by immunoblot analysis in a time-course experiment performed on MDA-MB 468, HepG2, LNCaP, and MCF-7 cells treated with beta-HCH (10 muM). Actin was used as normalization protein.   Figure 7 Immunoblot analysis of HIF-1alpha expression in MDA-MB 468, HepG2, LNCaP, and MCF-7 cells after 8 h treatment with 10 muM beta-HCH. Analysis was performed after 8 h treatment with 10 muM beta-HCH, as well as on cells pre-incubated with a specific STAT3 inhibitor (S3I-201) before beta-HCH treatment. Actin was used as normalization protein.   Figure 5 Microglial gene expression and function 6 months after immune stimulation. a, Weighted gene correlation network analysis (top: correlation coefficient; bottom: P-value; n=9,9,6,6,5,4 animals). b , Selected KEGG pathways enriched in modules. c , Heatmaps of genes within modules, z-scores (boxplot whiskers: 5-95 th percentile; n=1601,990,949,3543 genes in modules) and selected genes. d , Microglial mitochondrial membrane potential (left/middle; n=9,6,6,8,3,4 animals) and Pearson\'s correlation with lactate release (right; n=11,10,10 animals). e , Staining for top : HIF-1alpha, microglia (CD11b) and amyloid plaques (Methoxy-X04) and bottom : HIF-1alpha and microglial nuclei (Pu.1; single confocal plane) in brain sections from 9-month-old animals. Scale bars: 20/5 um (top/bottom). f , Total cellular (n=7,7,7 animals) and nuclear (n=8,8,7 animals) HIF-1alpha staining intensity. g , Microglial Abeta content (n=5,11,10,10 animals). */**/*** P   Figure 3. Hypoxia controls Gal-1 expression in KS through HIF-independent, NF-kappaB-dependent mechanisms. (A-D) Expression of Gal-1 in KS cells transfected with or without HIF-1alpha siRNA or a super-repressor form of IkappaB-alpha (IkappaB-alpha-SR) and incubated under hypoxia or normoxia. (A) Promoter activity. (B) qRT-PCR of Gal-1 mRNA relative to RN18S1. AU, arbitrary units. Data are the mean +- SEM of five (A) or three (B) independent experiments. (C) Immunoblot of Gal-1, IkappaB-alpha, HIF-1alpha, and actin. Data are representative of four independent experiments. (D) ELISA of Gal-1 secretion. Data are the mean +- SEM of three independent experiments. (E) ELISA of Gal-1 secretion by KS cells cultured under hypoxic or normoxic conditions in the presence or absence of HIF-1alpha or NF-kappaB inhibitors. Data are the mean +- SEM of three independent experiments. (F and G) Immunoblot (F) and qRT-PCR (G) of Gal-1 expression induced by hypoxia (Hyp) in human and mouse melanoma (A375 and B16-F0), mouse breast carcinoma (4T1), and human prostate carcinoma (LNCaP) cell lines. Data are representative (F) or are the mean +- SEM (G) of three independent experiments. (H) Western blot of HIF-1alpha, IkappaB-alpha, Gal-1, and actin upon treatment of KS cells with CoCl 2 (chemical activator of HIF-1alpha). Data are representative of four experiments. (I) Gal-1 promoter activity upon treatment of KS cells with CoCl 2 . Modulation of pGL3-Gal-1-Luciferase activity relative to Renilla ex   Figure 1 Effect of Cadmium on HIF-1alpha accumulation and hypoxia preconditioning protection. A : Cells were incubated under normoxia with 200 muM CoCl 2 (as a positive control), 5 muM CdCl 2 alone, hypoxia (0.5% O 2 ) alone, or hypoxia with CdCl 2 for 4 h. Whole cell lysates were collected immediately after treatment, separated on SDS-PAGE gel and blotted for HIF-1alpha. beta-actin was detected as a loading control. B : Cells were pretreated with hypoxia with or without CdCl 2 for 4 h and irradiated 2 min with UV. Four hours after irradiation, cells were fixed and stained for TUNEL. Bar graph shows percentage of TUNEL positive cells in indicated groups. Error bars represent standard error of the mean (n=3); the asterisk indicates significantly different from UV control (p   Figure 2 Effect of HIF-1alpha siRNA on HIF-1alpha protein expression. Cells were transfected with 15 and 25 nM HIF-1alpha siRNA and an siRNA non targeting control as indicated for 6 h. Twenty-four hours after transfection, cells were exposed to 4 h of hypoxia. Protein was collected immediately after treatment and blotted for HIF-1alpha. Bar graph represents the band intensity relative to the control. Error bars represent the standard error of the mean (n=3). beta-actin is detected as loading control.   Figure 5 Effect of HIF-1alpha siRNA on hypoxia-induced VEGF expression. Bovine stromal cells were transfected with HIF-1alpha siRNA or non-targeting siRNA control for 6 h. Twenty-four hours after transfection cells were preconditioned with hypoxia for 4 h. A : Total RNA was collected immediately after treatment. Image shows RT-PCR analysis for HIF-1alpha and VEGF . 18s rRNA was detected as an internal control. B : Protein analysis of VEGF and HIF-1alpha. Whole cell lysates were collected immediately after treatment, separated by SDS PAGE and probed for VEGF and HIF-1alpha. beta-actin was detected as a loading control. Bar graph shows VEGF expression in indicated groups relative to control. Error bars represent standard error of the mean (n=3). MA1-516 detects hypoxia-inducible factor 1 alpha (HIF-1 alpha) from human, non-human primate, bovine, mouse and porcine cells. This antibody does not cross-react with ARNT or the related HIF-2 alpha.rrMA1-516 has been successfully used in Western blot, immunofluorescence, immunoprecipitation, immunocytochemistry, and gel shift procedures. By Western blot, this antibody detects an ~116 kDa protein representing HIF-1 alpha after hypoxic induction in COS cells. Immunofluorescence staining of HIF-1 alpha in COS-7 cells with MA1-516 yields nuclear staining after exposing cells to 1% oxygen for 4 hours. In gel shift assays, MA1-516 has been successfully used only with mouse HIF-1 alpha.rrAntibodies to this protein (and modification) were previously sold as part of a Thermo Scientific Cellomics High Content Screening Kit. This replacement antibody is now recommended for researchers who need an antibody for high content cell based assays. It has been thoroughly tested and validated for cellular immunofluorescence (IF) applications. Further optimization including the selection of the most appropriate fluorescent Dylight conjugated secondary antibody may have to be performed for your high content assay. HIF1-alpha (HIF1A) is a subunit of HIF1, which is a transcription factor found in mammalian cells cultured under reduced oxygen tension. HIF-1 is a heterodimer consisting of an alpha and beta subunit, both belonging to the basic-helix-loop-helix Per-aryl hydrocarbon receptor nuclear translocator-Sim (PAS) family of transcription factors. HIF1 functions as a transcriptional regulator of the adaptive response to hypoxia. Under hypoxic conditions, HIF-1 activates the transcription of over 40 genes, including erythropoietin, glucose transporters, glycolytic enzymes, vascular endothelial growth factor, HILPDA, and other genes whose protein products increase oxygen delivery or facilitate metabolic adaptation to hypoxia. HIF1-alpha regulates hypoxia-mediated apoptosis, cell proliferation and tumor angiogenesis. Hypoxia which induces p53 protein accumulation, directly interacts with HIF1-alpha and reduces hypoxia-induced expression of HIF1-alpha by promoting MDM2-mediated ubiquitination and proteasomal degradation under hypoxic conditions. Recent studies suggest that induction of NOX4 by HIF1-alpha contributes to maintain ROS levels after hypoxia and hypoxia-induced proliferation. In humans, it is located on the q arm of chromosome 14. The C-terminal of HIF1A binds to p300. p300/CBP-HIF complexes participate in the induction of hypoxia-responsive genes, including VEGF. Hypoxia contributes significantly to the pathophysiology of major categories of human disease, including myocardial and cerebral ischemia, cancer, pulmonary hypertension, congenital heart disease and chronic obstructive pulmonary disease. 蛋白别名: ARNT interacting protein; ARNT-interacting protein; ARNT2; Basic-helix-loop-helix-PAS protein MOP1; bHLHe78; Class E basic helix-loop-helix protein 78; HIF; hif 1; hif 1a; HIF-1-alpha; HIF1-alpha; hypoxia inducible factor 1 alpha subunit; hypoxia inducible factor 1, alpha subunit (basic helix-loop-helix transcription factor); hypoxia-inducible factor 1 alpha; hypoxia-inducible factor 1 alpha isoform I.3; hypoxia-inducible factor 1, alpha subunit (basic helix-loop-helix transcription factor); Hypoxia-inducible factor 1-alpha; hypoxia-inducible factor1alpha; Member of PAS protein 1; member of PAS superfamily 1; PAS domain-containing protein 8 基因别名: AA959795; BHLHE78; HIF-1-alpha; HIF-1A; HIF-1alpha; HIF1; HIF1-ALPHA; HIF1A; HIF1alpha; MOP1; PASD8 Host server : magellan-srch-3-prod-green:8080/10.253.228.100:8080. git-commit: 2cd8645d2fc6bfe4ccb4abfa14772b0a94f68e98 git-url: http://victoria.invitrogen.com:8333/magellan/core.git git-branch: origin/release/1.27.0-2021.08.32-1.0

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