Preparation and characterization of monoclonal antibodies against mouse Jab1/CSN5 protein.
Jab1, also known as the fifth component of the COP9 signalosome complex (CSN5), directly interacts with and regulates the activity and stability of multiple intracellular regulatory molecules, such as c-Jun, p27, p53, Cullin, Smad4, and HIF1alpha. In addition, a high level of Jab1 is observed in a variety of human cancers and is sometimes correlated with a poor prognosis, suggesting that Jab1 contributes to cancer cell proliferation and survival and could be a novel target of cancer therapy. In this report, we generated five mouse monoclonal antibodies to a bacterially produced recombinant mouse Jab1 protein and examined their capabilities and limitations in commonly used assays, including enzyme linked immunosorbent assay (ELISA), Western blotting with denatured and native polyacrylamide gel electrophoresis (PAGE), immunoprecipitation, and immunofluorescence microscopy, finding the most suitable antibody for each application. Because these antibodies proved useful for immunohistochemical staining for Jab1 in fixed sections of human cancer samples, they should be useful in determining the expression and subcellular distribution of Jab1 in human tumors.
Kato
Jun-Ya
JY
Graduate School of Biological Sciences, Nara Institute of Science and Technology, Nara, Japan. jkata@bs.naist.jp
Nakamae
Ikuko
I
Tomoda
Kiichiro
K
Fukumoto
Akihisa
A
Yoneda-Kato
Noriko
N
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000302:isolation & purification; D000075686:COP9 Signalosome Complex; D002460:Cell Line; D006801:Humans; D006825:Hybridomas / Q000276:immunology; D007150:Immunohistochemistry; D047908:Intracellular Signaling Peptides and Proteins / Q000276:immunology* / Q000378:metabolism; D020014:K562 Cells; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D041681:NIH 3T3 Cells; D010190:Pancreatic Neoplasms / Q000378:metabolism; D010447:Peptide Hydrolases / Q000276:immunology* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D047908:Intracellular Signaling Peptides and Proteins; D010447:Peptide Hydrolases; C501490:Cops5 protein, mouse; D000075686:COP9 Signalosome Complex