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Complementary methods provide evidence for the expression of CXCR 7 on human B cells
Author(s) -
Humpert MarieLuise,
Tzouros Manuel,
Thelen Sylvia,
Big Alexandre,
Levoye Angélique,
ArenzanaSeisdedos Fernando,
Balabanian Karl,
Bachelerie Françoise,
Langen Hanno,
Thelen Marcus
Publication year - 2012
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.201100581
Subject(s) - chemokine , biology , chemokine receptor , microbiology and biotechnology , monoclonal antibody , transfection , epitope , antibody , receptor , cc chemokine receptors , cell culture , immunology , biochemistry , genetics
PTMs of extracellular domains of membrane proteins can influence antibody binding and give rise to ambivalent results. Best proof of protein expression is the use of complementary methods to provide unequivocal evidence. CXCR 7, a member of the atypical chemokine receptor family, mainly functions as scavenger for the chemokines CXCL 12 and CXCL 11. The expression of CXCR 7 on nonhematopoietic cells and neoplasms is widely accepted, however, its expression on leukocytes was recently challenged. To solve the dissent, we thoroughly analyzed the expression of CXCR 7 on human B cells. We validated the efficiency of different epitope‐specific monoclonal antibodies to detect CXCR 7 on transfected cells and primary human B cells. The specificity of the used antibodies was further confirmed by an experimentally independent double labeling approach. Examination of CXCR 7‐dependent scavenging of fluorescent‐labeled CXCL 12 revealed functional expression of the receptor on human B cells. Moreover, real‐time PCR analysis of CXCR 7 m RNA showed the presence of transcripts in human leukocytes. Finally, two CXCR 7‐specific peptides were identified by MS in immunoprecipitates from primary human B cells. Thus, we present a strategy based on combined proteomic and functional approaches that can be used to solve dissents on protein expression, i.e. demonstrating the expression of CXCR 7 on human leukocytes.