title: Progesterone receptor or cytoskeletal protein?
abstract: Immunoblotting is used to characterize the various nuclear progesterone receptor (nPR) isoforms present in tissues; however, the success of this technique is dependent on the specificity of the primary nPR antibody. The authors investigate the specificity of a frequently used nPR antibody, sc-538, in total protein from human myometrium and a myometrial cell line (PHM1-31). Using immunoblotting, 2 sc-538 immunoreactive bands at 100 and 55 kDa were detected. The bands were extracted and identified by 1-dimensional liquid chromatography mass spectrometry. The predominant protein in the 100-kDa band was alpha-actinin. The dominant proteins in the smaller band were vimentin (57 kDa) and desmin (53 kDa). Myometrial lysate was immunoprecipitated with sc-538, and immunoblotting of the immunoprecipitate with antibodies to alpha-actinin, desmin, and vimentin confirmed the presence of these proteins. The sc-538 nPR antibody therefore cross-reacts with cytoskeletal proteins that could be misinterpreted as nPR isoforms. Such misinterpretation has confused the progesterone response literature.
authors.affiliation: Mothers and Babies Research Centre, Hunter Medical Research Institute, University of Newcastle, John Hunter Hospital, Newcastle, NSW, Australia. gemma.madsen@newcastle.edu.au
authors.forename: Gemma
authors.initials: G
authors.lastname: Madsen
authors.forename: David A
authors.initials: DA
authors.lastname: Macintyre
authors.forename: Sam
authors.initials: S
authors.lastname: Mesiano
authors.forename: Roger
authors.initials: R
authors.lastname: Smith
mesh_terms: D000199:Actins / Q000302:isolation & purification*; D000328:Adult; D000906:Antibodies / Q000276:immunology*; D000918:Antibody Specificity; D002460:Cell Line; D002853:Chromatography, Liquid; D003598:Cytoskeletal Proteins / Q000276:immunology*; D003893:Desmin / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D009215:Myometrium / Q000737:chemistry / Q000276:immunology*; D011247:Pregnancy; D011980:Receptors, Progesterone / Q000276:immunology*; D020539:Sequence Analysis, Protein; D014746:Vimentin / Q000302:isolation & purification
chemical_list: D000199:Actins; D000906:Antibodies; D003598:Cytoskeletal Proteins; D003893:Desmin; D011980:Receptors, Progesterone; D014746:Vimentin; C523553:sc-538 antibody, human