TY - JOUR
T1 - Functional and immunochemical characterisation of different antibodies against the erythropoietin receptor
AU - Kirkeby, Agnete
AU - van Beek, Johan
AU - Nielsen, Jacob
AU - Leist, Marcel
AU - Helboe, Lone
PY - 2007/8/15
Y1 - 2007/8/15
N2 - Since it was discovered that the hematopoietic hormone erythropoetin (EPO) exerts neuroprotective effects in the CNS, many studies on the EPO receptor (EPOR) function and localisation in the CNS have been performed. For this purpose, commercially available anti-EPOR antibodies have often been applied. As the literature data on these antibodies show inconsistencies, we here systematically compared six frequently used, commercially available EPOR antibodies for different applications. Five of the antibodies appeared to specifically recognize recombinant rat and human EPOR in HEK293 cells by Western blotting, but the same antibodies yielded different and inconsistent results when human UT-7 cells or rat brain tissue were applied. Immunocytochemical staining of EPOR-transfected HEK cells only produced consistent results with three of the six antibodies. All antibodies stained neurons in rat brain sections, but with large differences in the staining pattern and only the C-20 EPOR antibody was found to label astrocytes. Since EPOR antibodies have been applied in several studies as EPOR antagonists, we further tested the antibodies for their capacity to functionally block the EPO-EPOR interaction in a cellular signalling system with STAT-5 phosphorylation as readout. Here, only the MAB307 antibody showed a partial effect at concentrations of 5-50 μg/ml.
AB - Since it was discovered that the hematopoietic hormone erythropoetin (EPO) exerts neuroprotective effects in the CNS, many studies on the EPO receptor (EPOR) function and localisation in the CNS have been performed. For this purpose, commercially available anti-EPOR antibodies have often been applied. As the literature data on these antibodies show inconsistencies, we here systematically compared six frequently used, commercially available EPOR antibodies for different applications. Five of the antibodies appeared to specifically recognize recombinant rat and human EPOR in HEK293 cells by Western blotting, but the same antibodies yielded different and inconsistent results when human UT-7 cells or rat brain tissue were applied. Immunocytochemical staining of EPOR-transfected HEK cells only produced consistent results with three of the six antibodies. All antibodies stained neurons in rat brain sections, but with large differences in the staining pattern and only the C-20 EPOR antibody was found to label astrocytes. Since EPOR antibodies have been applied in several studies as EPOR antagonists, we further tested the antibodies for their capacity to functionally block the EPO-EPOR interaction in a cellular signalling system with STAT-5 phosphorylation as readout. Here, only the MAB307 antibody showed a partial effect at concentrations of 5-50 μg/ml.
KW - Antibodies
KW - Brain
KW - EPOR antagonist
KW - Erythropoietin receptor
KW - Immunoblotting
KW - Immunohistochemistry
UR - http://www.scopus.com/inward/record.url?scp=34250622423&partnerID=8YFLogxK
U2 - 10.1016/j.jneumeth.2007.03.026
DO - 10.1016/j.jneumeth.2007.03.026
M3 - Journal article
C2 - 17524492
AN - SCOPUS:34250622423
SN - 0165-0270
VL - 164
SP - 50
EP - 58
JO - Journal of Neuroscience Methods
JF - Journal of Neuroscience Methods
IS - 1
ER -