Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes

Peled, Michael; Strazza, Marianne; Mor, Adam

doi:10.3791/58433

Cited by 1 publication

(2 citation statements)

References 16 publications

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“…The first two trials are designated by the month in which the AP was conducted (April or January). For the third trial, phosphatase inhibitor was added to the lysis buffer, which is often used in addition to protease inhibitors to potentially help stabilize extracted receptor protein complexes . This experiment is designated as phosphatase inhibitor (PhoIn).…”

Section: Resultsmentioning

confidence: 99%

“…For the third trial, phosphatase inhibitor was added to the lysis buffer, which is often used in addition to protease inhibitors to potentially help stabilize extracted receptor protein complexes. 57 This experiment is designated as phosphatase inhibitor (PhoIn). The three experimental trials also differed slightly in the centrifugal speed at which the aphid homogenate was clarified following tissue solubilization.…”

Section: Technical Assessment Of Sequential Ap−ms Experimental Trialsmentioning

confidence: 99%

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Affinity Purification–Mass Spectrometry Identifies a Novel Interaction between a Polerovirus and a Conserved Innate Immunity Aphid Protein that Regulates Transmission Efficiency

et al. 2021

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The vast majority of plant viruses are transmitted by insect vectors, with many crucial aspects of the transmission process being mediated by key protein–protein interactions. Still, very few vector proteins interacting with viruses have been identified and functionally characterized. Potato leafroll virus (PLRV) is transmitted most efficiently by Myzus persicae, the green peach aphid, in a circulative, non-propagative manner. Using affinity purification coupled to high-resolution mass spectrometry (AP–MS), we identified 11 proteins from M. persicaedisplaying a high probability of interaction with PLRV and an additional 23 vector proteins with medium confidence interaction scores. Three of these aphid proteins were confirmed to directly interact with the structural proteins of PLRV and other luteovirid species via yeast two-hybrid. Immunolocalization of one of these direct PLRV-interacting proteins, an orthologue of the human innate immunity protein complement component 1 Q subcomponent-binding protein (C1QBP), shows that MpC1QBP partially co-localizes with PLRV in cytoplasmic puncta and along the periphery of aphid gut epithelial cells. Artificial diet delivery to aphids of a chemical inhibitor of C1QBP leads to increased PLRV acquisition by aphids and subsequently increased titer in inoculated plants, supporting a role for C1QBP in the acquisition and transmission efficiency of PLRV by M. persicae. This study presents the first use of AP–MS for the in vivo isolation of a functionally relevant insect vector–virus protein complex. MS data are available from ProteomeXchange.org using the project identifier PXD022167.

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Section: Resultsmentioning

confidence: 99%

Section: Technical Assessment Of Sequential Ap−ms Experimental Trialsmentioning

confidence: 99%

Affinity Purification–Mass Spectrometry Identifies a Novel Interaction between a Polerovirus and a Conserved Innate Immunity Aphid Protein that Regulates Transmission Efficiency

et al. 2021

View full text Add to dashboard Cite

show abstract

Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes

Cited by 1 publication

References 16 publications

Affinity Purification–Mass Spectrometry Identifies a Novel Interaction between a Polerovirus and a Conserved Innate Immunity Aphid Protein that Regulates Transmission Efficiency

Affinity Purification–Mass Spectrometry Identifies a Novel Interaction between a Polerovirus and a Conserved Innate Immunity Aphid Protein that Regulates Transmission Efficiency

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