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

Abstract

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. IntroductionErythropoietin (Epo) is a low-molecular weight glycoprotein produced in the kidney by neural peritubular fibroblasts and by the outer medulla (1,2), depending on the amount of oxygen available (3). The Epo hormone facilitates the creation and maturation of erythroid precursors, and stimulates erythropoiesis upon binding to its receptor, known as erythropoietin receptor (EpoR) (4). Recombinant human Epo (rhEpo) is currently available for the treatment of anemia resulted from chronic liver diseases (5) or chronic renal failure (6), and is a good alternative to blood transfusion in cancer-related anemia (7), a frequent symptom of cancer (8) due to chemotherapy (9).The presence of EpoR has been confirmed in numerous tumor cells, with or without the stimulatory effect of Epo on these cells (10-16). While Epo/EpoR signaling in hematopoietic cells is associated with increased cell proliferation and/or survival, the Epo/EpoR signaling pathway in tumor cells does not always lead to increased cell proliferation, but may alter the sensitivity of cancer cells to therapy (17). The role of Epo and EpoR on growth, survival and therapeutic response in human cancer cells has been previously reviewed by Szenajch et al (17) and Debeljak et al (18).In this regard, although certain studies did not confirm a direct stimulatory effect of Epo on tumor cells, there is sufficient evidence of this effect on endothelial cell proliferation and/or tumor angiogenesis. Epo induced angiogenesis in murine hepatic tumors (19)

Mycoplasma genitalium Protein of Adhesion Promotes the Early Proliferation of Human Urothelial Cells by Interacting with RPL35

“…The interaction between rMgPa and RPL35 was identified from total cell proteins using far-Western blotting as described by Fecková et al [46]. Briefly, 10 µg of rMgPa was separated by 12% of SDS-PAGE and transferred to a PVDF membrane using a semi-dry transfer unit.…”