title: In Situ Proximity Ligation Assay (In Situ PLA) to Assess PTP-Protein Interactions.
abstract: Spatiotemporal aspects of protein-tyrosine phosphatase (PTP) activity and interaction partners for many PTPs are elusive. We describe here an elegant and relatively simple method, in situ proximity ligation assay (in situ PLA), which can be used to address these issues. The possibility to detect endogenous unmodified proteins in situ and to visualize individual interactions with spatial resolution is the major advantage of this technique. We provide protocols suitable to monitor association of the transmembrane PTPs PTPRJ/DEP-1/CD148 and PTPRB/VE-PTP with their substrates, the receptor tyrosine kinases FMS-like tyrosine kinase 3 (FLT3/CD135), and Tie2 and vascular endothelial growth factor receptor 2 (VEGFR2), respectively. Detailed description of method development and reagents as well as highlighting of critical factors will enable the reader to apply the method successfully to other PTP-protein interactions.
authors: Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.|Department 2, Max-Planck-Institute for Molecular Physiology, Dortmund, Germany.
authors: Sina
authors: S
authors: Koch
authors: Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.|Alere Technologies GmbH, Jena, Germany.
authors: Irene
authors: I
authors: Helbing
authors: Institute of Molecular Cell Biology, CMB, Jena University Hospital, Hans-Knöll-Strasse 2, 07745, Jena, Germany.
authors: Sylvia-Annette
authors: SA
authors: Böhmer
authors: Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.|Department of Physiology and Cell Biology, Graduate School of Medicine, Kobe University, Kobe, Hyogo, Japan.
authors: Makoto
authors: M
authors: Hayashi
authors: Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.
authors: Lena
authors: L
authors: Claesson-Welsh
authors: Department of Immunology, Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.|Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Biomedical Center, Uppsala University, Uppsala, Sweden.
authors: Ola
authors: O
authors: Söderberg
authors: Institute of Molecular Cell Biology, CMB, Jena University Hospital, Hans-Knöll-Strasse 2, 07745, Jena, Germany. boehmer@med.uni-jena.de.
authors: Frank-D
authors: FD
authors: Böhmer
mesh_terms: D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D057809:HEK293 Cells; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D061848:Optical Imaging / Q000379:methods; D025941:Protein Interaction Mapping / Q000379:methods*; D060066:Protein Interaction Maps; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D042787:Receptor, TIE-2 / Q000378:metabolism; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism; D051941:fms-Like Tyrosine Kinase 3 / Q000378:metabolism
chemical_list: D042787:Receptor, TIE-2; D040301:Vascular Endothelial Growth Factor Receptor-2; D051941:fms-Like Tyrosine Kinase 3; D017027:Protein Tyrosine Phosphatases
keywords: Imaging; In situ proximity ligation assay (in situ PLA); Protein interaction; Protein-tyrosine phosphatase; Receptor tyrosine kinase; Signal transduction