Monoclonal antibodies against Xenopus greatwall kinase.
Mitosis is known to be regulated by protein kinases, including MPF, Plk1, Aurora kinases, and so on, which become active in M-phase and phosphorylate a wide range of substrates to control multiple aspects of mitotic entry, progression, and exit. Mechanistic investigations of these kinases not only provide key insights into cell cycle regulation, but also hold great promise for cancer therapy. Recent studies, largely in Xenopus, characterized a new mitotic kinase named Greatwall (Gwl) that plays essential roles in both mitotic entry and maintenance. In this study, we generated a panel of mouse monoclonal antibodies (MAbs) specific for Xenopus Gwl and characterized these antibodies for their utility in immunoblotting, immunoprecipitation, and immunodepletion in Xenopus egg extracts. Importantly, we generated an MAb that is capable of neutralizing endogenous Gwl. The addition of this antibody into M-phase extracts results in loss of mitotic phosphorylation of Gwl, Plk1, and Cdk1 substrates. These results illustrate a new tool to study loss-of-function of Gwl, and support its essential role in mitosis. Finally, we demonstrated the usefulness of the MAb against human Gwl/MASTL.
Department of Oral Biology, College of Dentistry, University of Nebraska Medical Center, Lincoln, Nebraska, USA.
Ling
L
Wang
Laura A
LA
Fisher
James K
JK
Wahl
Aimin
A
Peng
D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived / Q000096:biosynthesis* / Q000737:chemistry; D057134:Antibodies, Neutralizing / Q000096:biosynthesis* / Q000737:chemistry; D000918:Antibody Specificity; D015153:Blotting, Western; D002457:Cell Extracts / Q000737:chemistry; D045744:Cell Line, Tumor; D005260:Female; D006801:Humans; D047468:Immunoprecipitation; D000090204:Mesothelin; D051379:Mice; D008807:Mice, Inbred BALB C; D008869:Microtubule-Associated Proteins / Q000096:biosynthesis / Q000737:chemistry; D009865:Oocytes / Q000201:enzymology; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D014981:Xenopus; D029867:Xenopus Proteins / Q000037:antagonists & inhibitors / Q000276:immunology* / Q000302:isolation & purification
D058846:Antibodies, Monoclonal, Murine-Derived; D057134:Antibodies, Neutralizing; D002457:Cell Extracts; D008869:Microtubule-Associated Proteins; C000715528:Msln protein, mouse; D011994:Recombinant Proteins; D029867:Xenopus Proteins; C544010:MASTL protein, Xenopus; C555237:MASTL protein, human; D017346:Protein Serine-Threonine Kinases; D000090204:Mesothelin