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Complementary methods provide evidence for the expression of CXCR7 on human B cells.

Proteomics (2012-05-25)
Marie-Luise Humpert, Manuel Tzouros, Sylvia Thelen, Alexandre Bignon, Angélique Levoye, Fernando Arenzana-Seisdedos, Karl Balabanian, Françoise Bachelerie, Hanno Langen, Marcus Thelen
ABSTRACT

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. CXCR7, a member of the atypical chemokine receptor family, mainly functions as scavenger for the chemokines CXCL12 and CXCL11. The expression of CXCR7 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 CXCR7 on human B cells. We validated the efficiency of different epitope-specific monoclonal antibodies to detect CXCR7 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 CXCR7-dependent scavenging of fluorescent-labeled CXCL12 revealed functional expression of the receptor on human B cells. Moreover, real-time PCR analysis of CXCR7 mRNA showed the presence of transcripts in human leukocytes. Finally, two CXCR7-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 CXCR7 on human leukocytes.

MATERIALS
Product Number
Brand
Product Description

Supelco
Atto 565 NHS ester, BioReagent, suitable for fluorescence, ≥70.0% (coupling to amines)
Sigma-Aldrich
Atto 565, BioReagent, suitable for fluorescence, ≥75% (sum of isomers, HPCE/HPLC)
Sigma-Aldrich
Atto 565-Biotin, BioReagent, suitable for fluorescence, ≥90% (HPCE)
Sigma-Aldrich
Atto 565 maleimide, BioReagent, suitable for fluorescence
Sigma-Aldrich
Atto 565 azide, BioReagent, suitable for fluorescence, ≥80.0% (HPCE)
Sigma-Aldrich
Atto 565 iodoacetamide, BioReagent, suitable for fluorescence

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