Characterization of HPC-1 antigen, an isoform of syntaxin-1, with the isoform-specific monoclonal antibody, 14D8.
We raised polyclonal and monoclonal antibodies against rat recombinant HPC-1/syntaxin 1A lacking a transmembrane domain. The polyclonal antibody recognized two major bands at 35 and 40 kDa from rat brain membranes. A hybridoma clone designated 14D8, however, recognized only one band at 35 kDa. A polyclonal antibody detected recombinant syntaxin 1B, as well as HPC-1/syntaxin 1A on an immunoblot, whereas 14D8 recognized recombinant HPC-1/ syntaxin 1A, but not syntaxin 1B. Therefore, 14D8 is specific for HPC-1/syntaxin 1A. Using this monoclonal antibody, we investigated the expression of HPC-1/syntaxin 1A in the rat hippocampal membranes. HPC-1/syntaxin 1A was present even in the embryonic d 19 (E19) hippocampal membranes, and it increased during the next two postnatal wk. Pyramidal cell axons were intensely stained with the 14D8 monoclonal antibody, suggesting that HPC-1/syntaxin 1A was not restricted to the presynaptic terminal. Furthermore, we investigated the phosphorylation of HPC-1/syntaxin 1A in the rat brain membranes. HPC-1/syntaxin 1A affinity-purified on a 14D8 IgG-coupled column was recognized by antiphosphoserine antibody, but not by antiphosphotyrosine and phosphothreonine antibodies.
Kushima
Y
Y
Department of Physiology, Kyorin University, School of Medicine, Tokyo, Japan.
Fujiwara
T
T
Sanada
M
M
Akagawa
K
K
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D000918:Antibody Specificity; D000954:Antigens, Surface / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D002540:Cerebral Cortex / Q000737:chemistry; D018507:Gene Expression Regulation, Developmental / Q000502:physiology; D006624:Hippocampus / Q000737:chemistry; D007074:Immunoglobulin G / Q000737:chemistry; D051379:Mice; D008807:Mice, Inbred BALB C; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D016716:PC12 Cells / Q000737:chemistry; D010766:Phosphorylation; D011817:Rabbits; D051381:Rats; D011919:Rats, Inbred Strains; D012160:Retina / Q000737:chemistry; D016622:Silver Staining; D050827:Syntaxin 1
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D007074:Immunoglobulin G; D009419:Nerve Tissue Proteins; C494214:Stx1a protein, mouse; C494213:Stx1a protein, rat; D050827:Syntaxin 1