Hypoxia inducible factor 1 (HIF-1) is a protein that plays an essential role in hypoxia, or low levels of cellular oxygen. HIF-1 is a heterodimeric protein that consists of a constitutively expressed beta subunit and oxygen related alpha subunit. Both subunits have a basic helix-loop-helix domain that leads to dimerization, where HIF-1 alpha carries an oxygen-dependent degradation (ODD) domain. Outside of the role of HIF-1 in hypoxia adaptation, it is also involved in cancer, inflammation and metabolism. The HIF-1 alpha subunit is specifically degraded in normal oxygen environments, where it is not active and functioning until a hypoxic environment is sensed. In some cases, HIF-1 alpha expression can be quite high, leading to angiogenesis and increased blood flow. The link between HIF-1 alpha activity and retinal pathways has long been established, given that photoreceptors in the retina require a large amount of oxygen to function properly. The use of a HIF-1 alpha antibody in the research of HIF-1 alpha signaling and behavior in the retina is highlighted below.
-Western%20Blot-NB100-105-img0019.jpg)
Western Blot: HIF-1 alpha Antibody (H1alpha67) [NB100-105] - Analysis using the HRP conjugate of NB100-105. Detection of 50ug cobalt chloride induced COS-7 nuclear extracts (NB800-PC26) using NB100-105.
To begin, Paeng et al used a HIF-1 alpha antibody to help elucidate the role of caffeic acid phenethyl ester (CAPE) in vascular endothelial growth factor reduction in the human retinal pigment epithelium under hypoxic conditions. First, a HIF-1 alpha antibody was used in western blot on ARPE-19 cells that had been treated with CAPE. This experiment determined that translocation of HIF-1 alpha to the nucleus in hypoxic ARPE-19 cells was nearly two-fold of that in normoxic conditions. However, with pretreatment of CAPE, the translocation of HIF-1 alpha to the nucleus was greatly prevented. The HIF-1 alpha antibody was also used in western blot to test the effects of a known number of inhibitory signaling transduction pathways. Interestingly, AKT had an increase in expression after 2 hours of increased hypoxia, however pre-treatment with CAPE reduced hypoxia-induced AKT phosphorylation. Overall, it was determined that HIF-1 alpha translocates to the nucleus, binds to the hypoxia response element of VEGF, and leads to ocular angiogenesis.
Next, Park et al used a HIF-1 antibody to show that 3,3’-Diindolylmethane (DIM) inhibits VEGF expression through HIF-1 alpha and NF-κB pathways in the retinal epithelium in hypoxic conditions. For starters, the HIF-1 alpha antibody was used in western blot analysis of retinal pigment epithelial (RPE) cells to show that HIF-1 alpha translocates to the nucleus under hypoxic conditions, which is the foundational step of hypoxia experiments. Subsequently, the interaction and binding of HIF-1 alpha and NF-κB was solidified through an electrophoretic mobility shift assay. A HIF-1 alpha antibody was also used to show that translocation of HIF-1 alpha and NF-κB in this model was halted by introduction to CoCl2 treatment. Ultimately it was hypothesized that pre-treatment of RPE cells in hypoxic conditions with a ROS inhibitor, caused a down regulation of VEGF through the inhibition of HIF-1 alpha.
Novus Biologicals offers HIF-1 alpha reagents for your research needs including:
PMID: 25738890
PMID: 25955241
PMID: 18160990
