Hypoxia Signaling: HIF1-alpha & HIF2-alpha Recombinant Rabbit Monoclonal Antibodies

The hypoxic response pathway is triggered by low levels of oxygen in the cellular environment. Hypoxia inducible transcription factor (HIF) is central to the hypoxic response. HIF exists as a heterodimeric transcription factor composed of an alpha and beta subunit. In mammals there are three HIF-alpha subunits, HIF1-alpha, HIF2-alpha, and HIF3-alpha, and one beta subunit, the aryl hydrocarbon receptor nuclear translocator (ARNT). The overexpression of HIF1-alpha and HIF2-alpha is associated with poor survival rates for various cancers. Experimental and clinical evidence strongly suggests HIF1-alpha and HIF2-alpha influence tumor development and response to treatment. Because of this, there has been major interest in developing selective HIF inhibitors; but due to the complexity of the HIF pathway, the process has been challenging. Thus, future work for therapeutic targeting of the HIFs will require a better understanding of both the HIF1-alpha and HIF2-alpha pathways.

Bethyl has expanded its capabilities and now offers rabbit recombinant monoclonal antibodies to HIF1-alpha and HIF2-alpha. These antibodies are available as a trial size (marked in the catalog ending in "-T") and have been validated for multiple applications.

Detection of Human HIF1-alpha by Immunohistochemistry

Detection of Human HIF1-alpha by Immunohistochemistry. Sample: FFPE section of prostate carcinoma. Antibody: Rabbit anti-Hif1-alpha recombinant monoclonal antibody (BL-124-3F7) A700-001 (lot A700-001-1) used for immunohistochemistry at 1:100. Detection: DAB.

Detection of Human HIF2-alpha by Western Blot of Immunoprecipitates
Detection of Human HIF2-alpha by Western Blot of Immunoprecipitates. Samples: Whole cell lysate (1 mg for IP, 20% of IP loaded) from HEPG2 cells that were treated with 200 µM cobalt chloride (+) or mock treated (-). Antibody: Rabbit anti-HIF2-alpha recombinant monoclonal antibody [BL-95-1A2] A700-003 (lot number A700-003-2) used at 20 µl per reaction. For blotting immunoprecipitated HIF2-alpha, A700-003 was used at 1:1000. Detection: Chemiluminescence with exposure time of 3 minutes.
Localization of HIF1 alpha Binding Sites by ChIP-sequencing

Localization of HIF1 alpha Binding Sites by ChIP-sequencing. Chromatin from CoCl2 treated HepG2 cells was immunoprecipitated with anti-HIF1 alpha antibody A700-001 and analyzed by DNA sequencing. The figure illustrates the peak distribution of HIF1 alpha binding within a 250 Kb region of chromosome 11 as detected using anti-HIF1 alpha antibody A700-001. ChIP-seq validation performed by Active Motif, Carlsbad, CA.

Trial sizes are cost-effective and ideal for comparison testing.
Trial size antibodies are ideal for comparisons between suppliers or various applications testing, and are cost effective. More than 6,000 trial-sized antibodies are currently available to over 3,300 protein targets, and more are being validated every month.


70% of the nearly 64,000 cases of kidney cancers diagnosed each year in the U.S. are classified as clear-cell renal carcinoma (ccRCC). In ccRCC, the von Hippel-Lindau tumor suppressor gene (VHL) is the most frequently mutated gene. VHL loss in ccRCC is described as a "truncal" event: an early lesion that conspires with later genetic hits to lead to kidney cancer. When VHL is absent, HIF2-alpha accumulates, resulting in the activation of transcriptional programs accommodating angiogenesis and the adaptation of cells to hypoxia. An understanding of this mechanism has led to the discovery of a number of drugs that target HIF2-alpha effectors. For example, there are currently at least six drugs approved by the FDA targeting VEGF or its receptor. Despite the proven efficacy of these drugs, the clinical response to these agents has been found to be temporary as tumor cells can become resistant.

If HIF2-alpha could be targeted directly, it may be possible to cast a wider net to inhibit multiple transcriptional and signaling programs led by HIF2-alpha-responsive genes. Since transcription factors are viewed as undruggable, this concept has been deemed unapproachable. However, the recent discovery of a pocket in the PAS-B domain of HIF2-alpha responsible for mediating the dimerization between HIF2-alpha and its obligatory partner, ARNT, has led to the idea of designing small molecular drugs that could prevent dimerization, and thus antagonize HIF2-alpha transcriptional activity. Scientists at Peloton Therapeutics in Dallas, Texas have discovered a class of small molecular compounds able to antagonize HIF2-alpha transcriptional programs by inhibiting HIF2-alpha-ARNT dimerization. In vitro studies as well as studies in xenograft ccRCC models conducted at Peloton and in two academic labs have evaluated the anti-tumor efficacy of these compounds. The studies showed that one of these small-molecule drugs effectively inhibited tumor progression. Furthermore, the most promising data comes from the study by Chen et al., which showed a patient with metastatic ccRCC remained free of progression for more than 11 months after treatment with the small-molecule drug. The next steps will require the discovery of predictive biomarkers to identify patients that would be responsive to treatments aimed at dismantling HIF2 transcriptional programs and further validation of HIF2-alpha as a target for the treatment of ccRCC.

Bethyl's Comparison

Bethyl's Comparison'

Antibodies against HIF2-alpha were assessed for specificity by immunocytochemistry (ICC). HIF2-alpha protein is rapidly degraded under normoxic conditions and stabilized under hypoxic conditions. Cobalt chloride treatment can be used as a mimetic agent for hypoxia to stimulate HIF2-alpha stabilization. Antibodies against HIF2-alpha were compared by staining HepG2 cells treated with cobalt chloride or mock-treated.

The antibodies included a Bethyl recombinant rabbit monoclonal antibody (Catalog # A700-003), a widely available mouse monoclonal, clone EP190b, from Competitor A, and a goat polyclonal from Competitor B. As expected, in untreated cells, no staining was observed for all antibodies except the Competitor B goat polyclonal, which exhibited off-target nuclear staining. In treated cells, upregulated, nuclear localized, HIF2-alpha was correctly detected by Bethyl’s Cat# A700-003. This was in contrast to Competitor A’s antibody which detected an upregulated protein incorrectly localized to the cytoplasm, and Competitor B’s antibody which detected no change in response to treatment.

*All antibodies were tested as per supplier datasheets.

HepG2 untreated and HepG2 treated with CoCl2
Detection of human HIF2-alpha by immunocytochemistry in HepG2 cells treated without cobalt chloride (left) and with cobalt chloride (right). Antibody: Rabbit anti-HIF2-alpha recombinant monoclonal antibody (BL-95-1A2), Bethyl Cat# A700-003. Cobalt chloride treatment served as a mimetic agent for hypoxia to activate HIF2-alpha stabilization.

To view Bethyl's complete Hypoxia portfolio, click here.


Patel PH, Chadalavada RS, Chaganti RS, Motzer RJ. 2006. Targeting von Hippel-Lindau pathway in renal cell carcinoma. Clin Cancer Res. Dec 15;12(24):7215-20.

Cho H, Kaelin WG. 2016. Targeting HIF2 in clear cell renal cell carcinoma. Cold Spring Harb Symp Quant Biol 81: 113-121.

Wallace EM, Rizzi JP, Han G, Wehn PM, Cao Z, Du X, Cheng T, Czerwinski RM, Dixon DD, Goggin BS, et al. 2016. A small-molecule antagonist of HIF2α is efficacious in preclinical models of renal cell carcinoma. Cancer Res. Sep 15;76(18):5491-500.

Chen W, Hill H, Christie A, Kim MS, Holloman E, Pavia-Jimenez A, Homayoun F, Ma Y, Patel N, Yell P, et al. 2016. Targeting renal cell carcinoma with a HIF-2 antagonist. Nature. Nov 3;539(7627):112-117.

Cho H, Du X, Rizzi JP, Liberzon E, Chakraborty AA, Gao W, Carvo I, Signoretti S, Bruick RK, Josey JA, et al. 2016. On-target efficacy of a HIF-2α antagonist in preclinical kidney cancer models. Nature. Nov 3;539(7627):107-111.