Development of polyclonal antibodies specific to ATP-binding cassette transporters human ABCG4 and mouse Abcg4: site-specific expression of mouse Abcg4 in brain.
In our recent study on seeking new mouse ATP-binding cassette (ABC) transporters of the G subfamily, we succeeded in cloning mouse Abcg4 from a cDNA library of mouse brain, and we characterized the tissue-specific expression and chromosomal localization of the mouse Abcg4 gene. To further characterize the physiological function of mouse Abcg4 protein and to compare its function with that of ABCG2, in the present study, we developed polyclonal antibodies against mouse Abcg4 and established the Abcg4-expression system. To raise antibodies, we selected three different epitope peptides that correspond to the amino acid residues of 46-60, 465-479, and 600-613 in mouse Abcg4 protein. The antibody raised against the epitope encoding the amino acids 46-60 was found to be specific to mouse Abcg4, exhibiting a band with molecular weight of 63,000 on immunoblotting, whereas this band was dose-dependently diminished by adding the corresponding epitope peptide into the immunoblot medium. Use of the antibody for immunoblot detection in mouse normal tissues revealed that the Abcg4 protein is expressed in brain, spleen, and testis. Immunohistochemical studies showed that mouse Abcg4 is site-specifically expressed in the cerebral cortex and medulla of mouse brain. These results suggest that mouse Abcg4 plays a certain physiological role in the brain. It is of importance to note that the sequence of amino acids 46-60 is completely identical between mouse Abcg4 and human ABCG4. Thus, this antibody is applicable to the detection of human ABCG4 as well as mouse Abcg4.
Department of Biomolecular Engineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama 226-8501, Japan.
Shoko
S
Koshiba
Takehito
T
Ito
Akira
A
Shiota
Kanako
K
Wakabayashi
Masatsugu
M
Ueda
Hiroshi
H
Ichinose
Toshihisa
T
Ishikawa
D000070996:ATP Binding Cassette Transporter, Subfamily G; D018528:ATP-Binding Cassette Transporters / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D000595:Amino Acid Sequence; D016564:Amyloid beta-Protein Precursor / Q000378:metabolism; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D001923:Brain Chemistry; D000939:Epitopes; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology; D018411:Spodoptera
C439766:ABCG4 protein, human; D000070996:ATP Binding Cassette Transporter, Subfamily G; D018528:ATP-Binding Cassette Transporters; C486685:Abcg4 protein, mouse; D016564:Amyloid beta-Protein Precursor; D000906:Antibodies; D000939:Epitopes; D011994:Recombinant Proteins