In Situ Detection of Phosphorylated Platelet-derived Growth Factor Receptor β 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 ␤ (PDGFR␤) 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 PDGFR␤ in human embryonic kidney cells stably overexpressing human influenza hemagglutinin-tagged human PDGFR␤ in porcine aortic endothelial cells transfected with the ␤-receptor, but not in cells transfected with the ␣-receptor, and also in immortalized human foreskin fibroblasts, BJ hTert, endogenously expressing the PDGFR␤. We furthermore visualized tyrosine phosphorylated PDGFR␤ 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. Molecular & Cellular Proteomics 6:1500 -1509, 2007.

BlobFinder, a tool for fluorescence microscopy image cytometry

“…PDFGF-BB stimulates the phosphorylation of platelet-derived growth factor receptorˇ(PDGFRˇ). PDGFRˇphosphorylation was detected by in situ PLA, resulting in bright point like signals [1]. Ten images were acquired for each concentration 0, 1, 3, 10, 30 and 100 ng/ml of PDGF-BB (see Fig.…”

“…There are many different methods for in situ detection of sub-cellular structures, e.g., immunofluorescence staining, in situ Proxim-ity Ligation Assay (in situ PLA) [1], FISH (fluorescent in situ hybridization) and padlock-probing [2]. Although the procedures for these methods differ, the images produced share many similarities.…”

In Situ Analysis of Mutant EGFRs Prevalent in Glioblastoma Multiforme Reveals Aberrant Dimerization, Activation, and Differential Response to Anti-EGFR Targeted Therapy

“…EGFR phosphoactivation analysis was conducted using mouse anti-EGFR (1:400; Upstate) and rabbit anti-pEGFR1068 (1:400; Cell Signalling). In situ PLA was conducted following primary antibody incubation as described previously (10,11). PLA probe dilution/incubation time and rolling circle amplification (RCA) times were all optimized for this specific application.…”

Mammary-derived growth inhibitor (MDGI) interacts with integrin α-subunits and suppresses integrin activity and invasion

“…This technique visualizes two proteins in close proximity by detecting a fluorescent signal produced by the rolling-circle amplification reaction (Jarvius et al, 2007). Cells are stained with primary antibodies, followed by incubation with oligolinked secondary antibodies.…”