Generation and characterization of novel human IRAS monoclonal antibodies.
Imidazoline receptors were first proposed by Bousquet et al., when they studied antihypertensive effect of clonidine. A strong candidate for I1R, known as imidazoline receptor antisera-selected protein (IRAS), has been cloned from human hippocampus. We reported that IRAS mediated agmatine-induced inhibition of opioid dependence in morphine-dependent cells. To elucidate the functional and structure properties of I1R, we developed the newly monoclonal antibody against the N-terminal hIRAS region including the PX domain (10-120aa) through immunization of BALB/c mice with the NusA-IRAS fusion protein containing an IRAS N-terminal (10-120aa). Stable hybridoma cell lines were established and monoclonal antibodies specifically recognized full-length IRAS proteins in their native state by immunoblotting and immunoprecipitation. Monoclonal antibodies stained in a predominantly punctate cytoplasmic pattern when applied to IRAS-transfected HEK293 cells by indirect immunofluorescence assays and demonstrated excellent reactivity in flow immunocytometry. These monoclonal antibodies will provide powerful reagents for the further investigation of hIRAS protein functions.
Wang
Bo
B
Department of Pharmacology, Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China.
Liu
Ying
Y
Shan
Yajun
Y
Yao
Zhenyu
Z
Liu
Xiaolan
X
Su
Ruibin
R
Sun
Qihong
Q
Cong
Yuwen
Y
Li
Jin
J
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000276:immunology / Q000378:metabolism; D002460:Cell Line; D004926:Escherichia coli / Q000235:genetics; D005434:Flow Cytometry; D006801:Humans; D006825:Hybridomas / Q000378:metabolism; D054755:Imidazoline Receptors / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D015151:Immunoblotting; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism
D000911:Antibodies, Monoclonal; D054755:Imidazoline Receptors; D047908:Intracellular Signaling Peptides and Proteins; C458505:NISCH protein, human; D011993:Recombinant Fusion Proteins