Hypoxia contributes significantly to the pathophysiology of major categories of human disease, including myocardial and cerebral ischemia, cancer, pulmonary hypertension, congenital heart disease and chronic obstructive pulmonary disease. HIF-1 is a nuclear protein involved in mammalian oxygen homeostasis. This occurs as a posttranslational modification by prolyl hydroxylation. HIF-1 is a heterodimer composed of HIF-1 alpha and HIF-1 beta subunits. Both subunits are constantly translated. However, under normoxic conditions, human HIF-1 alpha is hydroxylated at Pro402 or Pro564 by a set of HIF prolyl hydroxylases, is polyubiquinated, and eventually degraded in proteosomes. Under hypoxic conditions, the lack of hydroxylation prevents HIF degradation and increases transcriptional activity. Therefore, the concentration of HIF-1 alpha increases in the cell. In contrast, HIF-1 beta remains stable under either condition. HIF hydroxylases provide insight into hypoxic cell responses, which may be used to help isolate therapeutic targets.
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Western Blot: HIF-1 alpha Antibody (H1alpha67) - On day 1, MEF cells (+/+,-/-), were grown on 15 cm dish (2x10 to the 6th cells). On day 2, cells were exposed to hypoxia for 4hrs. Cells were washed with ice cold PBS twice and whole cell protein was extracted with RIPA buffer fortified with protease. Upon quantification, 100ug of protein was fractionated on 7% polyacralymide gel. -Gel was transferred overnight onto nitrocellulose membrane. The membrane was probed with HIF-1 alpha monoclonal antibody at a 1:500 dilution . The secondary antibody was conjugated with HRP and was used at a 1:2500 dilution. Photo courtesy of Dr. Gregg Semenza, Johns Hopkins University.