- PA1-184 - Invitrogen Antibodies HIF1A antibody

Submitted references VHL regulates the sensitivity of clear cell renal cell carcinoma to SIRT4-mediated metabolic stress via HIF-1α/HO-1 pathway.

Tong Y, Kai J, Wang S, Yu Y, Xie S, Zheng H, Wang Y, Liu Y, Zhu K, Guan X, Guo L, Lu R
Cell death & disease 2021 Jun 16;12(7):621

LncRNA TUG1 contributes to the tumorigenesis of lung adenocarcinoma by regulating miR-138-5p-HIF1A axis.

Li K, Niu H, Wang Y, Li R, Zhao Y, Liu C, Cao H, Chen H, Xie R, Zhuang L
International journal of immunopathology and pharmacology 2021 Jan-Dec;35:20587384211048265

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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 20 µg of HeLa whole cell lysate either untreated (Lane 2) or treated with 100 µm deferoxamine for 16 hours (Lane 3) and 7 µL of Molecular Weight Protein Ladder per well onto a 4-12% Bis-Tris polyacrylamide gel. Proteins were transferred to a nitrocellulose membrane and blocked with 5% BSA/TBST for at least 1 hour. The membrane was probed with a anti-HIF1-alpha polyclonal antibody (Product # PA1-184) at a dilution of 1:1000 overnight at 4°C on a rocking platform, washed in TBS-0.1%Tween-20, and probed with Clean-blot IP detection reagent (Product # 21230) at a dilution of 1:2000 for at least 1 hour. Chemiluminescent detection was performed using SuperSignal West Dura (Product # 34075).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of HIF1 alpha (green) in untreated HeLa cells (Figure A) and HeLa cells treated with 100 µm Deferoxamine Mesylate for ~16 hours (Figure B). 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 Anti-HIF1 alpha polyclonal antibody (Product # PA1-184) at a dilution of 1:100 for at least 1 hour at room temperature, washed with PBS, and incubated with DyLight 488 goat anti-rabbit IgG secondary antibody (Product # 35552) 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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of HIF-1 alpha was done on 70% confluent log phase MCF-7 cells treated with 0.6 nM of CoCl2 for 6 hours. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled HIF-1 alpha Rabbit Polyclonal Antibody (Product # PA1-184) at 2µg/mL in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 for 45 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® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d is a merged image showing nuclear localization. Panel e shows untreated cells with cytoplasmic signal. Panel f represents no primary antibody control. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunoprecipitation of HIF1-alpha was performed on HeLa cells that were either untreated (Lane 2) or treated with 100 µm deferoxamine for 16 hours (Lane 3). Antigen-antibody complexes were formed by incubating 750 µg of HeLa whole cell lysate with 2 µg of an anti-HIF1-alpha polyclonal antibody (Product # PA1-184) overnight on a rocking platform at 4øC. The immune complexes were captured on 100 µL Protein A/G Plus Agarose (Product # 20423), washed extensively, and eluted with reducing sample loading dye. Samples were resolved on a 4-12% Bis-Tris polyacrylamide gel, transferred to a nitrocellulose membrane, and blocked with 5% BSA/TBS-0.1%Tween for at least 1 hour. The membrane was probed with PA1-184 at a dilution of 1:1000, washed in TBST, and probed with Clean-blot IP detection reagent (Product # 21230) at a dilution of 1:2000 for at least 1 hour. Chemiluminescent detection was performed using SuperSignal West Dura (Product # 34075).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 6 SIRT4 interacts with HIF-1alpha and directly suppresses the expression of HIF-1alpha. A Analyzed the correlation between HIF-1alpha and SIRT4 in mutated VHL and nonmutated VHL group from a TCGA cohort of ccRCC patients. B HIF-1alpha protein in SIRT4 overexpressed 786-O and Caki-2 cells were displayed by western blot. The quantification analysis is shown on the right panel. C Caki-2 cells were treated with VHL inhibitor, VH-298 (50 uM), for 24 h followed by western blot. Densitometric analyses of HIF-1alpha/HO-1 are shown on the right panel. D Molecular docking of SIRT4 and HIF-1alpha was realized by zdock. E The immunoprecipitation was used to analysis the interaction of exogenous SIRT4 with endogenous HIF-1alpha.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 6. miR-138-5p reversed TUG1-induced HIF1A expression in LAC cells. A , The binding sites between miR-138-5p and WT or Mut HIF1A 3'UTR. B, C, The luciferase activity of WT HIF1A 3'UTR was repressed by miR-138-5p mimics, but that of Mut HIF1A 3'UTR was unaffected by miR-138-5p mimics in H1975 and A549. D, E , qRT-PCR and Western blot analysis of the HIF1A expression levels after co-transfection with sh-TUG1 lentiviruses and miR-138-5p inhibitors in H1975 and A549 cell lines. * p

PA1-184

Antibody data