Generation and characterization of monoclonal antibodies that specifically recognize p65/L-plastin isoform but not T-plastin isoform.
The 65-kDa protein (p65) was previously identified as a phosphorylated protein in activated macrophages, and has turned out to be a member of a plastin protein family characterized by a series of Ca(2+)-, calmodulin-, and beta-actin-binding domains. In mice, two isoforms, p65/L-plastin and T-plastin, have so far been identified; p65/L-plastin is expressed in hemopoietic cells and cancer cells, and T-plastin in solid tissue cells. We generated monoclonal antibodies to p65/L-plastin, examined the isoform-specificity by using recombinant (r) T-plastin, and found that the antibodies were specific for rp65/L-plastin, whereas immune sera to rp65/L-plastin showed cross-reactions to rT-plastin. One of the antibodies, p65-7B5, was demonstrated to react to native p65/L-plastin by Western blot, flow cytometric, and immunohistochemical analysis. Furthermore, p65-7B5 has made it possible to detect p65/L-plastin-expressing cells in tissues where T-plastin is abundantly expressed. These reagents and procedures should provide specific tools to investigate the role of p65/L-plastin in leukocytes.
Toyooka
Kiminori
K
International Medical Center of Japan, Tokyo. hiroto@m.ehime-u.ac.jp
Liu
Fenzhi
F
Ishii
Motoshi
M
Saito
Shouichiro
S
Kirikae
Teruo
T
Asano
Yoshihiro
Y
Shinomiya
Hiroto
H
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology*; D000918:Antibody Specificity / Q000276:immunology*; D002460:Cell Line; D007150:Immunohistochemistry; D008562:Membrane Glycoproteins; D051379:Mice; D008840:Microfilament Proteins; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D020033:Protein Isoforms / Q000737:chemistry / Q000276:immunology
D000911:Antibodies, Monoclonal; D008562:Membrane Glycoproteins; D008840:Microfilament Proteins; D010750:Phosphoproteins; D020033:Protein Isoforms; C026742:plastin