Hypoxia Pathway Antibody Sampler Kit

The Hypoxia Pathway Antibody Sampler Kit provides an economical means to investigate select proteins involved in the hypoxia pathway. The kit contains enough primary antibodies to perform two western blot experiments with each primary antibody.

Hypoxia-inducible factor 1 (HIF1) is a heterodimeric transcription factor that plays a critical role in the cellular response to hypoxia (1). The HIF1 complex consists of two subunits, HIF-1α and HIF-1β, which are basic helix-loop-helix proteins of the PAS (Per, ARNT, Sim) family (2). HIF1 regulates the transcription of a broad range of genes that facilitate responses to the hypoxic environment, including genes regulating angiogenesis, erythropoiesis, cell cycle, metabolism, and apoptosis. The widely expressed HIF-1α is typically degraded rapidly in normoxic cells by the ubiquitin/proteasomal pathway. Under normoxic conditions, HIF-1α is proline hydroxylated leading to a conformational change that promotes binding to the von Hippel-Lindau protein (VHL) E3 ligase complex; ubiquitination and proteasomal degradation follows (3,4). Both hypoxic conditions and chemical hydroxylase inhibitors (such as desferrioxamine and cobalt) inhibit HIF-1α degradation and lead to its stabilization. In addition, HIF-1α can be induced in an oxygen-independent manner by various cytokines through the PI3K-AKT-mTOR pathway (5-7).

HIF-1β is also known as AhR nuclear translocator (ARNT) due to its ability to partner with the aryl hydrocarbon receptor (AhR) to form a heterodimeric transcription factor complex (8). Together with AhR, HIF-1β plays an important role in xenobiotics metabolism (8). In addition, a chromosomal translocation leading to a TEL-ARNT fusion protein is associated with acute myeloblastic leukemia (9). Studies also found that ARNT/HIF-1β expression levels decrease significantly in pancreatic islets from patients with type 2 diabetes, suggesting that HIF-1β plays an important role in pancreatic β-cell function (10).

Hypoxia-inducible factor (HIF) is essential for the cellular response to hypoxia (11,12). There are several isoforms of the HIF α subunit. Studies have found that HIF-1α and HIF-2α expression is increased in some human cancers. HIF-1α has both pro- and anti-proliferative activities, whereas HIF-2α does not possess anti-proliferative activity (12). Therefore, HIF-2α likely plays an important role in tumorigenesis (12,13).

FIH (Factor inhibiting HIF-1, HIF asparagine hydroxylase) is a dioxygen-dependent asparaginyl hydroxylase that modifies target protein function by hydroxylating target protein asparagine residues (14-16). HIF, a transcriptional activator involved in control of cell cycle in response to hypoxic conditions, is an important target for FIH regulation. FIH functions as an oxygen sensor that regulates HIF function by hydroxylating at Asn803 in the carboxy-terminal transactivation domain (CAD) of HIF (17,18). During normoxia, FIH uses cellular oxygen to hydroxylate HIF-1 and prevent interaction of HIF-1 with transcriptional coactivators, including the CBP/p300-interacting transactivator. Under hypoxic conditions, FIH remains inactive and does not inhibit HIF, allowing the activator to regulate transcription of genes in response to low oxygen conditions (17-19). FIH activity is regulated through interaction with proteins, including Siah-1, which targets FIH for proteasomal degradation (20). The Cut-like homeodomain protein CDP can bind the FIH promoter region to regulate FIH expression at the transcriptional level (21). Phosphorylation of HIF at Thr796 also can prevent FIH hydroxylation on Asn803 (22). Potential FIH substrates also include proteins with ankyrin repeat domains, such as Iκ-B, Notch, and ASB4 (23-25).

PHD1 (Egln2), PHD-2 (Egln1), and PHD3 (Egln3) are members of the Egln family of proline hydroxylases. They function as oxygen sensors that catalyze the hydroxylation of HIF on prolines 564 and 402, initiating the first step of HIF degradation through the VHL/ubiquitin pathway (26,27).