title: Issues associated with assessing nuclear localization of N-terminally unphosphorylated beta-catenin with monoclonal antibody 8E7.
abstract: BACKGROUND
  Beta-catenin is a dual function adhesion/transcriptional co-activator protein, and both functions are critical for normal tissue homeostasis. Since the transcriptional functions of beta-catenin are more often implicated in various disease processes, there is much interest in the development and use of reagents to interrogate spatial and temporal evidence of beta-catenin nuclear signaling in cells and tissues. An important study demonstrated that the signaling form of beta-catenin is specifically unphosphorylated at residues S37 and T41, and suggested that this form exhibits a propensity for cytosolic/nuclear accumulation relative to the total pool of beta-catenin.
  
  
  RESULTS
  We show that monoclonal antibody, 8E7, which recognizes the signaling form of beta-catenin specifically unphosphorylated at S37 and T41 (Active B-Catenin, ABC), also cross-reacts with a widely expressed, variably accessible nuclear antigen that is not beta-catenin. In cell types commonly used to study Wnt activation, this non-specific nuclear staining can be robust, obscuring the ABC signal. Definitive detection of nuclear localized ABC can be confirmed through an ability of classical cadherins to sequester ABC to cell junctions. In tissues, milder antigen retrieval methods can reduce the accessibility of mAb 8E7 to this cross-reacting nuclear antigen.
  
  
  CONCLUSIONS
  These findings reveal that interpretation of nuclear, signaling active beta-catenin using monoclonal antibody 8E7 should be considered judiciously, and in conjunction with independent methods.
authors.affiliation: Department of Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA. m-thorne@northwestern.edu
authors.forename: Meghan T
authors.initials: MT
authors.lastname: Maher
authors.forename: Annette S
authors.initials: AS
authors.lastname: Flozak
authors.forename: Alyssa M
authors.initials: AM
authors.lastname: Hartsell
authors.forename: Susan
authors.initials: S
authors.lastname: Russell
authors.forename: Rohinee
authors.initials: R
authors.lastname: Beri
authors.forename: Ofra N
authors.initials: ON
authors.lastname: Peled
authors.forename: Cara J
authors.initials: CJ
authors.lastname: Gottardi
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis / Q000378:metabolism*; D000941:Antigens; D015820:Cadherins / Q000378:metabolism; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D006801:Humans; D008168:Lung / Q000166:cytology / Q000378:metabolism; D051379:Mice; D010766:Phosphorylation; D020033:Protein Isoforms / Q000378:metabolism; D021381:Protein Transport; D051381:Rats; D015398:Signal Transduction; D013194:Staining and Labeling; D013347:Subcellular Fractions / Q000378:metabolism; D051176:beta Catenin / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D000941:Antigens; D015820:Cadherins; D020033:Protein Isoforms; D051176:beta Catenin