Far-western blotting as a solution to the non-specificity of the anti-erythropoietin receptor antibody

Fecková,Barbora; Kimáková,Patrícia; Ilkovičová,Lenka; Szentpéteriová,Erika; Debeljak,Nataša; Solárová,Zuzana; Sačková,Veronika; Šemeláková,Martina; Bhide,Mangesh; Solár,Peter

doi:10.3892/ol.2016.4782

Far-western blotting as a solution to the non-specificity of the anti-erythropoietin receptor antibody

Authors:
- Barbora Fecková
- Patrícia Kimáková
- Lenka Ilkovičová
- Erika Szentpéteriová
- Nataša Debeljak
- Zuzana Solárová
- Veronika Sačková
- Martina Šemeláková
- Mangesh Bhide
- Peter Solár
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Affiliations: Department of Cell Biology, Institute of Biology and Ecology, Faculty of Science, Pavol Jozef Šafárik University in Košice, SK-04154 Košice, Slovak Republic, Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, SI‑1000 Ljubljana, Slovenia, Institute of Pharmacology, Faculty of Medicine, Pavol Jozef Šafárik University in Košice, SK-04001 Košice, Slovak Republic, Laboratory of Biomedical Microbiology and Immunology, University of Veterinary Medicine, SK-04181 Košice, Slovak Republic
Published online on: June 24, 2016 https://doi.org/10.3892/ol.2016.4782
Pages: 1575-1580

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Abstract

The erythropoietin receptor (EpoR) is a member of the cytokine receptor family. The interaction between erythropoietin (Epo) and EpoR is important for the production and maturation of erythroid cells, resulting in the stimulation of hematopoiesis. The fact that EpoR was also detected in neoplastic cells has opened the question about the relevance of anemia treatment with recombinant Epo in cancer patients. Numerous studies have reported pro‑stimulating and anti‑apoptotic effects of Epo in cancer cells, thus demonstrating EpoR functionality in these cells. By contrast, a previous study claims the absence of EpoR in tumor cells. This apparent discrepancy is based, according to certain authors, on the use of non‑specific anti‑EpoR antibodies. With the aim of bypassing the direct detection of EpoR with an anti-EpoR antibody, the present authors propose a far‑western blot methodology, which in addition, confirms the interaction of Epo with EpoR. Applying this technique, the presence of EpoR and its interaction with Epo in human ovarian adenocarcinoma A2780 and normal human umbilical vein endothelial cells was confirmed. Furthermore, modified immunoprecipitation of EpoR followed by matrix‑assisted laser desorption/ionization‑time‑of‑flight mass spectrometry analysis confirmed a 57 kDa protein as a human Epo-interacting protein in both cell lines.

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