title: Generation and characterization of monoclonal antibodies against human Chibby protein.
abstract: Chibby (Cby) was originally identified as an antagonist of the Wnt/β-catenin signaling pathway. It physically interacts with the key co-activator β-catenin and inhibits β-catenin-mediated transcriptional activation. More recently, we demonstrated that Cby protein localizes to the base of motile cilia and is required for ciliogenesis in the respiratory epithelium of mice. To gain further insight into the physiological function of Cby, we developed mouse monoclonal antibodies (MAbs) against human Cby protein and characterized two Cby MAbs, designated 8-2 and 27-11, in depth. Western blot analysis revealed that 8-2 reacts with both human and mouse Cby proteins, whereas 27-11 is specific to human Cby. The epitopes of 8-2 and 27-11 were narrowed down to the middle portion (aa 49-63) and N-terminal region (aa 1-31) of the protein, respectively. We also determined their isotypes and found that 8-2 and 27-11 belong to IgG2a and IgG1 with κ light chains, respectively. Both MAbs can be employed for immunoprecipitation assays. Moreover, 8-2 detects endogenous Cby protein on Western blots, and marks the ciliary base of motile cilia in the murine lung and trachea as shown by immunofluorescence staining. These Cby MAbs therefore hold promise as useful tools for the investigation of Wnt signaling and ciliogenesis.
authors.affiliation: Graduate Program in Molecular and Cellular Pharmacology, Department of Pharmacological Sciences, SUNY at Stony Brook, Stony Brook, New York, USA.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Cyge
authors.forename: Victoria
authors.initials: V
authors.lastname: Fischer
authors.forename: Ken-Ichi
authors.initials: K
authors.lastname: Takemaru
authors.forename: Feng-Qian
authors.initials: FQ
authors.lastname: Li
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification; D002352:Carrier Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D002923:Cilia / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D018604:Epitope Mapping; D004926:Escherichia coli; D057809:HEK293 Cells; D006801:Humans; D006825:Hybridomas / Q000276:immunology / Q000378:metabolism; D007132:Immunoglobulin Isotypes / Q000032:analysis; D007145:Immunoglobulin kappa-Chains / Q000737:chemistry; D047468:Immunoprecipitation; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011485:Protein Binding / Q000276:immunology; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D020545:Respiratory Mucosa / Q000166:cytology / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D014132:Trachea / Q000166:cytology / Q000378:metabolism; D014162:Transfection; D051153:Wnt Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; C473750:CBY1 protein, human; D002352:Carrier Proteins; C517894:Chibby protein, mouse; D007132:Immunoglobulin Isotypes; D007145:Immunoglobulin kappa-Chains; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D051153:Wnt Proteins