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AB259779

Human EPAS1 (HIF-2-alpha) knockout A549 cell lysate

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EPAS1 KO cell lysate available now. KO validated by Next Generation Sequencing, Western blot. Free of charge wild type control included. Knockout achieved by CRISPR/Cas9 X = 1 bp insertion Frameshift = 99%.

View Alternative Names

Basic-helix-loop-helix-PAS protein MOP2, Class E basic helix-loop-helix protein 73, ECYT4, EPAS1_HUMAN, Endothelial PAS domain-containing protein 1, Endothelial pas domain protein 1, HIF-1-alpha-like factor, HIF-2-alpha, HIF2A, Hypoxia inducible factor 2 alpha subunit, Hypoxia-inducible factor 2-alpha, MOP 2, Member of PAS protein 2, Member of pas superfamily 2, PAS domain-containing protein 2, PASD2, bHLHe73

3 Images
Western blot - Human EPAS1 (HIF-2-alpha) knockout A549 cell lysate (AB259779)
  • WB

Lab

Western blot - Human EPAS1 (HIF-2-alpha) knockout A549 cell lysate (AB259779)

Lane 1 : Wild-type A549 untreated cell lysate 40 ug
Lane 2 : Wild-type A549 + DFO (1mM, 24 hours) cell lysate 40 ug
Lane 3 : EPAS knockout A549 untreated cell lysate 40 ug
Lane 4 : EPAS knockout A549 + DFO (1mM, 24 hours) cell lysate 40 ug
Lanes 1 - 4 : Merged signal (red and green). Green - ab109616 observed at 100 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.
ab109616 was shown to react with HIF-2-alpha in wild-type A549 cells in western blot with loss of signal observed in EPAS1 knockout cell line ab259774 (knockout cell lysate ab259779). A549 wild-type and EPAS1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween®) before incubation with ab109616 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at 1 μg/ml and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-HIF-2-alpha antibody (<a href='/en-us/products/primary-antibodies/hif-2-alpha-antibody-ab109616'>ab109616</a>) at 1 µg/mL

Lane 1:

Wild-type A549 untreated cell lysate at 40 µg

Lane 2:

Wild-type A549 + DFO (1mM, 24 hours) cell lysate at 40 µg

Lane 2:

Western blot - Human EPAS1 (HIF-2-alpha) knockout A549 cell line (<a href='/en-us/products/cell-lines/human-epas1-hif-2-alpha-knockout-a549-cell-line-ab259774'>ab259774</a>)

Lane 3:

EPAS knockout A549 untreated cell lysate at 40 µg

Lane 4:

EPAS knockout A549 + DFO (1mM, 24 hours) cell lysate at 40 µg

Predicted band size: 96 kDa

Observed band size: 100 kDa

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Western blot - Human EPAS1 (HIF-2-alpha) knockout A549 cell lysate (AB259779)
  • WB

Supplier Data

Western blot - Human EPAS1 (HIF-2-alpha) knockout A549 cell lysate (AB259779)

Lane 1 : Wild-type A549 Untreated (DFO Control) cell lysate 20 μg
Lane 2 : Wild-type A549 Treated DFO (1 mM, 24 h) cell lysate 20 μg
Lane 3 : EPAS1 knockout A549 Untreated (DFO Control) cell lysate 20 μg
Lane 4 : EPAS1 knockout A549 Treated DFO (1 mM, 24 h) cell lysate 20 μg
False colour image of Western blot : Anti-HIF-2-alpha antibody [OTI2G5] staining at 1/500 dilution, shown in black; Rabbit Anti-GAPDH antibody [EPR16891] (ab181602) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab157249 was shown to bind specifically to HIF-2-alpha. A band was observed at 100 kDa in treated wild-type A549 cell lysates with no signal observed at this size in EPAS1 knockout cell line ab259774 (knockout cell lysate ab259779). To generate this image, wild-type and EPAS1 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5 % BSA in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times before development with Optiblot (ECL reagent ab133456) and imaged with 20 seconds exposure time. Secondary antibodies used were HRP conjugated Goat anti-Mouse (H+L) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed (ab216777) at 1/20000 dilution.

All lanes:

Western blot - Anti-HIF-2-alpha antibody [OTI2G5] (<a href='/en-us/products/primary-antibodies/hif-2-alpha-antibody-oti2g5-ab157249'>ab157249</a>) at 1/500 dilution

Lane 1:

Wild-type A549 Untreated (DFO Control) cell lysate at 20 µg

Lane 2:

Wild-type A549 Treated DFO (1 mM, 24 h) cell lysate at 20 µg

Lane 2:

Western blot - Human EPAS1 (HIF-2-alpha) knockout A549 cell line (<a href='/en-us/products/cell-lines/human-epas1-hif-2-alpha-knockout-a549-cell-line-ab259774'>ab259774</a>)

Lane 3:

EPAS1 knockout A549 Untreated (DFO Control) cell lysate at 20 µg

Lane 4:

EPAS1 knockout A549 Treated DFO (1 mM, 24 h) cell lysate at 20 µg

Predicted band size: 96 kDa

Observed band size: 100 kDa

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Next Generation Sequencing - Human EPAS1 (HIF-2-alpha) knockout A549 cell lysate (AB259779)
  • NGS

Lab

Next Generation Sequencing - Human EPAS1 (HIF-2-alpha) knockout A549 cell lysate (AB259779)

X = 1 bp insertion

Key facts

Cell type

A549

Species or organism

Human

Tissue

Lung

Knockout validation

Next Generation Sequencing,Western blot

Mutation description

Knockout achieved by CRISPR/Cas9 X = 1 bp insertion Frameshift = 99%

Disease

Carcinoma

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Reactivity data

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Product details

Knockout cell lysate achieved by CRISPR/Cas9.

REACH authorisation
Abcam has not and does not intend to apply for the REACH Authorisation of customers' uses of products that contain European Authorisation list (Annex XIV) substances.
It is the responsibility of our customers to check the necessity of application of REACH Authorisation, and any other relevant authorisations, for their intended uses.

Lysate preparation: Our lysates are made using RIPA buffer to which we add a protease inhibitor cocktail and phosphatase inhibitor cocktail (ratio: 300:100:10). This means that the protein of interest is denatured. If you require a native form of the protein please use the live cell version. Please refer to our lysis protocol for further details on how our lysates are prepared.

User storage instructions: Lyophilizate may be stored at 4°C. After reconstitution, store at -20°C for short-term storage or -80°C for long-term storage.

This product is subject to limited use licenses from The Broad Institute, ERS Genomics Limited and Sigma-Aldrich Co. LLC, and is developed with patented technology. For full details of the licenses and patents please refer to our limited use license and patent pages.

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What's included?

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Properties and storage information

Gene name
EPAS1
Gene editing type
Knockout
Gene editing method
CRISPR technology
Knockout validation
Next Generation Sequencing, Western blot
Shipped at conditions
Ambient - Can Ship with Ice
Appropriate short-term storage conditions
-20°C
Appropriate long-term storage conditions
-20°C
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Supplementary information

This supplementary information is collated from multiple sources and compiled automatically.

HIF-2-alpha also known as hypoxia-inducible factor 2-alpha or EPAS1 plays a significant role in cellular response to oxygen levels. Mechanically HIF-2-alpha functions as a transcription factor that activates certain genes when oxygen is low. The protein typically weighs around 118 kDa. It is expressed in various tissues but particularly in endothelial cells and some neuronal tissues. These sites highlight its importance in organs requiring tight regulation of oxygen.
Biological function summary

HIF-2-alpha regulates the expression of genes involved in energy metabolism and angiogenesis. The protein forms a complex by dimerizing with the HIF-1-beta subunit which is necessary for transcriptional activity. Through this complex formation and activity it influences processes such as erythropoiesis and regulates factors like vascular endothelial growth factor (VEGF). Therefore HIF-2-alpha contributes to the adaptation of cells and tissues under hypoxic conditions.

Pathways

HIF-2-alpha engages in the hypoxia signaling pathway playing an essential part by modulating gene expression in response to low oxygen availability. This role impacts other proteins such as HIF-1-alpha sharing overlapping functions but with distinct target genes. Additionally HIF-2-alpha is involved in the mTOR pathway which influences cell growth and metabolism through its interaction with the nutrient-sensing regulatory pathway.

Researchers have linked HIF-2-alpha with conditions like renal cell carcinoma and pulmonary hypertension. The protein's dysregulation can lead to altered expression of angiogenic factors promoting tumor growth and survival. In particular its connection with VEGF exemplifies its role in these pathologies by facilitating aberrant blood vessel formation. Such interactions further highlight the potential of HIF-2-alpha as a therapeutic target in disease modulation.
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Quality control

STR analysis

CSF1PO, D13S317, D7S820, D5S818, TH01, D16S539, TPOX

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Cell culture

Biosafety level

EU: 1 US: 1

Adherent/suspension

Adherent

Gender

Male

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Product protocols

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Complementary Products

Primary Antibodies

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Cell Lines & Lysates

AB259774

Human EPAS1 (HIF-2-alpha) knockout A549 cell line

Advanced Validation

Western blot - Human EPAS1 (HIF-2-alpha) knockout A549 cell line (AB259774)

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Cell Lines & Lysates

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Hela-DFO treated (0.5mM, 24h) Nuclear Lysate

Western blot - Hela-DFO treated (0.5mM, 24h) Nuclear Lysate (AB180880)

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Primary Antibodies

AB179483

Anti-HIF-1 alpha antibody [EPR16897]

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Immunocytochemistry/ Immunofluorescence - Anti-HIF-1 alpha antibody [EPR16897] (AB179483)

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Product promise

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