HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) All Versions of this Article: M700166-MCP200v1 6/9/1500    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Glossary Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Jarvius, M. Articles by Söderberg, O. Search for Related Content PubMed PubMed Citation Articles by Jarvius, M. Articles by Söderberg, O. Social Bookmarking                      What's this?

Molecular & Cellular Proteomics 6:1500-1509, 2007.
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

---

Research

In Situ Detection of Phosphorylated Platelet-derived Growth Factor Receptor ß Using a Generalized Proximity Ligation Method^*

Malin Jarvius^,, Janna Paulsson^,¶, Irene Weibrecht^,, Karl-Johan Leuchowius, Ann-Catrin Andersson^||, Carolina Wählby^,**, Mats Gullberg^||, Johan Botling, Tobias Sjöblom, Boyka Markova, Arne Östman^¶,, Ulf Landegren and Ola Söderberg^,¶¶

From the Department of Genetics and Pathology, Rudbeck Laboratory, University of Uppsala, SE-75185 Uppsala, Sweden, ^¶ Department of Oncology-Pathology, Cancer Center Karolinska, R8:03, Karolinska Institutet, SE-17176 Stockholm, Sweden, ^|| Olink AB, Dag Hammarskjöldsväg 54 A, SE-75183 Uppsala, Sweden, ^** Centre for Image Analysis, Uppsala University, SE-75105 Uppsala, Sweden, and 3rd Medical Department, Hematology-Oncology, Johannes Gutenberg University, 55131 Mainz, Germany

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 ß (PDGFRß) 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 PDGFRß in human embryonic kidney cells stably overexpressing human influenza hemagglutinin-tagged human PDGFRß in porcine aortic endothelial cells transfected with the ß-receptor, but not in cells transfected with the -receptor, and also in immortalized human foreskin fibroblasts, BJ hTert, endogenously expressing the PDGFRß. We furthermore visualized tyrosine phosphorylated PDGFRß 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.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				M. H. Vermeer, R. van Doorn, R. Dijkman, X. Mao, S. Whittaker, P. C. van Voorst Vader, M.-J. P. Gerritsen, M.-L. Geerts, S. Gellrich, O. Soderberg, et al. Novel and Highly Recurrent Chromosomal Alterations in Sezary Syndrome Cancer Res., April 15, 2008; 68(8): 2689 - 2698. [Abstract] [Full Text] [PDF]