Protein biomarker validation via proximity ligation assays

Blokzijl, Andries; Nong, Rachel Yuan; Darmanis, Spyros; Hertz, Ellen; Landegren, Ulf; Kamali‐Moghaddam, Masood

doi:10.1016/j.bbapap.2013.07.016

Cited by 33 publications

(37 citation statements)

References 43 publications

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“…This weakness could be overcome by coupling with promiscuous biotin ligase assay, where the biotin ligase fused with a target protein covalently attaches biotin to the interacting partners, thereby enabling high-affinity capture of them through streptavidin-conjugated beads39. Proximity ligation assay is a tool to sensitively detect PPIs using oligonucleotide-conjugated antibodies and subsequent signal amplification by PCR4041. While these assays can entrap PPIs among endogenous proteins, cells need to be lysed or fixed before analysis.…”

Section: Discussionmentioning

confidence: 99%

Detecting protein–protein interactions based on kinase-mediated growth induction of mammalian cells

Mabe

Nagamune

Kawahara

2014

Sci Rep

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Detection of protein–protein interactions (PPIs) is important for understanding numerous processes in mammalian cells; however, existing PPI detection methods often give significant background signals. Here, we propose a novel PPI-detection method based on kinase-mediated growth induction of mammalian cells. In this method, target proteins are fused to the intracellular domain of c-kit (c-kit ICD) and expressed in interleukin-3-dependent mammalian cells. The PPI induces dimerization and activation of c-kit ICDs, which leads to cell growth in the absence of interleukin-3. Using this system, we successfully detected the ligand-dependent homo-interaction of FKBPF36V and hetero-interaction of FKBP and FRBT2098L, as well as the constitutive interaction between MDM2 and a known peptide inhibitor. Intriguingly, cells expressing high-affinity peptide chimeras are selected from the mixture of the cell populations dominantly expressing low-affinity peptide chimeras. These results indicate that this method can detect PPIs with low background levels and is suitable for peptide inhibitor screening.

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Section: Discussionmentioning

confidence: 99%

Detecting protein–protein interactions based on kinase-mediated growth induction of mammalian cells

Mabe

Nagamune

Kawahara

2014

Sci Rep

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“…The details of these are enumerated in preceding publications [22][23][24], as well as in the vendor website (www.olink.com). Namely, two primary antibodies conforming to the proteins of interest were hybridized on formalin-fixed sections for standardized time.…”

Section: Study Of Trask Protein Interactions By Plamentioning

confidence: 99%

Significance of Trask protein interactions in brain metastatic cohorts of lung cancers

Shang

Chen

et al. 2015

Tumor Biol.

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A class of adhesion protein that occurs in the membrane with both extracellular and intracellular domain and play vital role in maintaining multicellularity is TRASK, also called CUB-domain containing protein1, CD318 (CDCP1). Specifically, in the current study, documented aggressive grades of lung cancers and distant metastatic tissues were examined for protein interactions of Trask and compared with lung cancer variants in situ. The intracellular domain of Trask has the ability to undergo tyrosine phosphorylation and thereafter undergo increased genomic expression, as well as interact with cytoskeletal proteins in the cell periphery and other local signal transduction machinery to induce invadopodia formation and distant metastasis. We incorporated proximity ligation assay to examine protein interactions of Trask in metastatic lung cancer tissues and compare with advanced and low-grade lung cancers restricted to the primary site of origins. Here, we provide direct evidence that activated Trask, which is a phosphorylated form, binds with cytoskeletal proteins actin and spectrin. These interactions were not seen in locally growing lung cancer and cancer in situ. These interactions may be responsible for invadopodia formation and breaking free from a multicellular environment. Functional studies demonstrated interaction between Trask and the STOCs Orai1 and Stim1. Calcium release from internal stores was highest in metastatic lung cancers, suggesting this mechanism as an initial stimulus for the cells to respond chaotically to external growth factor stimulation, especially in aggressive metastatic variants of lung cancers. Recently, inhibitors of STOCs have been identified, and preclinical evidence may be obtained whether these drugs may be of benefit in preventing the deadly consequences of lung cancer.

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“…To demonstrate the interaction between PCSK9 and CD81, we performed in situ PLA. This new approach allows the identification of single interactions in their natural envi- ronment (42)(43)(44) and has been used successfully in recent studies on PCSK9 (45) and tetraspanin proteins (46). In our experiment, the PCSK9-LDLR interaction was used as a positive control, and transfection with an empty vector served as a negative control.…”

Section: Volume 290 • Number 38 • September 18 2015mentioning

confidence: 99%

Plasma Membrane Tetraspanin CD81 Complexes with Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) and Low Density Lipoprotein Receptor (LDLR), and Its Levels Are Reduced by PCSK9

Blanchet

Seidah

et al. 2015

Journal of Biological Chemistry

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Protein biomarker validation via proximity ligation assays

Cited by 33 publications

References 43 publications

Detecting protein–protein interactions based on kinase-mediated growth induction of mammalian cells

Detecting protein–protein interactions based on kinase-mediated growth induction of mammalian cells

Significance of Trask protein interactions in brain metastatic cohorts of lung cancers

Plasma Membrane Tetraspanin CD81 Complexes with Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) and Low Density Lipoprotein Receptor (LDLR), and Its Levels Are Reduced by PCSK9

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