Generation and characterization of novel monoclonal antibodies against murine and human TARSH proteins.
TARSH/Abi3bp was originally isolated as a novel target of NESH-SH3 by a two-hybrid yeast system. We have already identified murine TARSH (mTARSH) as a cellular senescence-related gene because of its robust induction in the early phase of mouse embryonic fibroblast cellular senescence. We have also revealed that the expression of this gene was dramatically reduced in human lung cancer cell lines and primary lung tumor, while it was predominantly expressed in normal conditions. This evidence suggests that TARSH is involved in both stress-induced senescence and prevention of cancer development; however, little is known about its molecular mechanisms. To reveal the further physiological function of this molecule, we established rat anti-TARSH monoclonal antibodies (MAb). Recombinant His-tagged partial mouse TARSH protein was expressed in Escherichia coli, affinity purified and used as an antigen to immunize rats. Hybridomas were screened by enzyme-linked immunosorbent assay, and we generated six stable hybridoma cell lines that produced antibody against murine TARSH protein, including three clones that represented cross-reactivity with human TARSH. We determined their isotypes and further examined capabilities or limitations in immunoblotting, immunoprecipitation, and immunofluorescence microscopy, realizing the most suitable antibody for each application. These MAbs should therefore be very useful tools for the study of TARSH expression and for following biological function in cellular senescence and tumor suppression.
Uekawa
Natsuko
N
Department of Mechanism of Aging, National Institute for Longevity Sciences, National Center for Geriatrics and Gerontology, Obu-City, Aichi, Japan.
Nishioka
Tomohisa
T
Terauchi
Kunihiko
K
Ohta
Satoshi
S
Sugimoto
Masataka
M
Shimada
Jun-Ichi
J
Maruyama
Mitsuo
M
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000737:chemistry*; D000918:Antibody Specificity; D002352:Carrier Proteins / Q000276:immunology*; D002460:Cell Line; D005260:Female; D006801:Humans; D006825:Hybridomas; D051379:Mice; D008819:Mice, Nude; D051381:Rats; D017208:Rats, Wistar; D018909:src Homology Domains / Q000276:immunology
C439569:ABI3BP protein, human; D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; C527603:TARSH protein, mouse