ab20654 detects a HIF-2-alpha specific band at ~96 kDa in both unstimulated-PC12 cytoplasmic lysate and hypoxia-treated PC12 nuclear lysate. We do not know the identity of the additional bands observed. However, the bands at ~70 kDa (this could be HIF3 alpha) and 220 kDa have been observed with other HIF-2-alpha antibodies (Kiettzmann et al, 2001; Biochem. J. 354, pp531–537).
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 1 µg/ml. Detects a band of approximately 96 kDa (predicted molecular weight: 96 kDa).
Use a concentration of 1 µg/ml.
1/2500. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
Transcription factor involved in the induction of oxygen regulated genes. Binds to core DNA sequence 5'-[AG]CGTG-3' within the hypoxia response element (HRE) of target gene promoters. Regulates the vascular endothelial growth factor (VEGF) expression and seems to be implicated in the development of blood vessels and the tubular system of lung. May also play a role in the formation of the endothelium that gives rise to the blood brain barrier. Potent activator of the Tie-2 tyrosine kinase expression. Activation seems to require recruitment of transcriptional coactivators such as CREBPB and probably EP300. Interaction with redox regulatory protein APEX seems to activate CTAD.
Expressed in most tissues, with highest levels in placenta, lung and heart. Selectively expressed in endothelial cells.
Defects in EPAS1 are the cause of erythrocytosis familial type 4 (ECYT4) [MIM:611783]. ECYT4 is an autosomal dominant disorder characterized by increased serum red blood cell mass, elevated hemoglobin concentration and hematocrit, and normal platelet and leukocyte counts.
In normoxia, is probably hydroxylated on Pro-405 and Pro-531 by EGLN1/PHD1, EGLN2/PHD2 and/or EGLN3/PHD3. The hydroxylated prolines promote interaction with VHL, initiating rapid ubiquitination and subsequent proteasomal degradation. Under hypoxia, proline hydroxylation is impaired and ubiquitination is attenuated, resulting in stabilization. In normoxia, is hydroxylated on Asn-847 by HIF1AN thus probably abrogating interaction with CREBBP and EP300 and preventing transcriptional activation. Phosphorylated on multiple sites in the CTAD. The iron and 2-oxoglutarate dependent 3-hydroxylation of asparagine is (S) stereospecific within HIF CTAD domains.
ICC/IF image of ab20654 stained rat PC12 cells. The cells were PFA fixed (10 min), permabilised in PBS-T (20 min) and incubated with the antibody (ab20654, 1µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to block non-specific protein-protein interactions. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red). DAPI was used to stain the cell nuclei (blue).
ab20654 staining HIF-2-alpha in Adrenal gland. The paraffin embedded human tissue was incubated with ab20654(1:2500 dilution) for 30 mins at room temperature. Antigen retrieval was performed by heat induction in citrate buffer pH 6. Ab20654 was tested in a tissue microarray (TMA) containing a wide range of normal and cancer tissues as well as a cell microarray consisting of a range of commonly used, well characterised human cell lines.
Wu L et al. Overexpression and correlation of HIF-2a, VEGFA and EphA2 in residual hepatocellular carcinoma following high-intensity focused ultrasound treatment: Implications for tumor recurrence and progression. Exp Ther Med13:3529-3534 (2017).
Read more (PubMed: 28587437) »
Prieto-Domínguez N et al. Melatonin enhances sorafenib actions in human hepatocarcinoma cells by inhibiting mTORC1/p70S6K/HIF-1a and hypoxia-mediated mitophagy. Oncotarget8:91402-91414 (2017).
Read more (PubMed: 29207653) »