title: In situ detection of phosphorylated platelet-derived growth factor receptor beta using a generalized proximity ligation method.
abstract: Improved methods are needed for in situ characterization of post-translational modifications in cell lines and tissues. For example, it is desirable to monitor the phosphorylation status of individual receptor tyrosine kinases in samples from human tumors treated with inhibitors to evaluate therapeutic responses. Unfortunately the leading methods for observing the dynamics of tissue post-translational modifications in situ, immunohistochemistry and immunofluorescence, exhibit limited sensitivity and selectivity. Proximity ligation assay is a novel method that offers improved selectivity through the requirement of dual recognition and increased sensitivity by including DNA amplification as a component of detection of the target molecule. Here we therefore established a generalized in situ proximity ligation assay to investigate phosphorylation of platelet-derived growth factor receptor beta (PDGFRbeta) in cells stimulated with platelet-derived growth factor BB. Antibodies specific for immunoglobulins from different species, modified by attachment of DNA strands, were used as secondary proximity probes together with a pair of primary antibodies from the corresponding species. Dual recognition of receptors and phosphorylated sites by the primary antibodies in combination with the secondary proximity probes was used to generate circular DNA strands; this was followed by signal amplification by replicating the DNA circles via rolling circle amplification. We detected tyrosine phosphorylated PDGFRbeta in human embryonic kidney cells stably overexpressing human influenza hemagglutinin-tagged human PDGFRbeta in porcine aortic endothelial cells transfected with the beta-receptor, but not in cells transfected with the alpha-receptor, and also in immortalized human foreskin fibroblasts, BJ hTert, endogenously expressing the PDGFRbeta. We furthermore visualized tyrosine phosphorylated PDGFRbeta in tissue sections from fresh frozen human scar tissue undergoing wound healing. The method should be of great value to study signal transduction, screen for effects of pharmacological agents, and enhance the diagnostic potential in histopathology.
authors.affiliation: Department of Genetics and Pathology, Rudbeck Laboratory, University of Uppsala, SE-75185, Uppsala, Sweden.
authors.forename: Malin
authors.initials: M
authors.lastname: Jarvius
authors.forename: Janna
authors.initials: J
authors.lastname: Paulsson
authors.forename: Irene
authors.initials: I
authors.lastname: Weibrecht
authors.forename: Karl-Johan
authors.initials: KJ
authors.lastname: Leuchowius
authors.forename: Ann-Catrin
authors.initials: AC
authors.lastname: Andersson
authors.forename: Carolina
authors.initials: C
authors.lastname: Wählby
authors.forename: Mats
authors.initials: M
authors.lastname: Gullberg
authors.forename: Johan
authors.initials: J
authors.lastname: Botling
authors.forename: Tobias
authors.initials: T
authors.lastname: Sjöblom
authors.forename: Boyka
authors.initials: B
authors.lastname: Markova
authors.forename: Arne
authors.initials: A
authors.lastname: Ostman
authors.forename: Ulf
authors.initials: U
authors.lastname: Landegren
authors.forename: Ola
authors.initials: O
authors.lastname: Söderberg
mesh_terms: D000199:Actins / Q000378:metabolism; D002460:Cell Line; D042783:Endothelial Cells / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D007136:Immunoglobulins / Q000737:chemistry; D007150:Immunohistochemistry / Q000379:methods; D007668:Kidney / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods*; D020797:Receptor, Platelet-Derived Growth Factor beta / Q000378:metabolism*; D015398:Signal Transduction; D014443:Tyrosine / Q000737:chemistry; D014945:Wound Healing
chemical_list: D000199:Actins; D007136:Immunoglobulins; D014443:Tyrosine; D020797:Receptor, Platelet-Derived Growth Factor beta