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Direct visualization of newly synthesized target proteins in situ

tom Dieck, Susanne and Kochen, Lisa and Hanus, Cyril and Heumüller, Maximilian and Bartnik, Ina and Nassim-Assir, Belquis and Merk, Katrin and Mosler, Thorsten and Garg, Sakshi and Bunse, Stefanie and Tirrell, David A. and Schuman, Erin M. (2015) Direct visualization of newly synthesized target proteins in situ. Nature Methods, 12 (5). pp. 411-414. ISSN 1548-7091. PMCID PMC4414919. https://resolver.caltech.edu/CaltechAUTHORS:20150205-174152503

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[img] PDF (Supplementary Figures 1–7 and Supplementary Table 1) - Supplemental Material
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[img] Image (JPEG) (Supplementary Figure 1: Incubation scheme for FUNCAT-PLA and controls for FUNCAT-PLA and Puro-PLA) - Published Version
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[img] Image (JPEG) (Supplementary Figure 2: Experimental system to test the FUNCAT-PLA dependence on protein synthesis and coincident presence of AHA incorporation and the protein of interest) - Supplemental Material
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[img] Image (JPEG) (Supplementary Figure 3: Selectivity and specificity of labeling newly synthesized proteins) - Supplemental Material
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[img] Image (JPEG) (Supplementary Figure 4: Pulse-labeling followed by FUNCAT-PLA reveals distinct spatial patterns for newly synthesized protein species in neurons) - Supplemental Material
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[img] Image (JPEG) (Supplementary Figure 5: Bassoon FUNCAT-PLA signal is present in axons, and comparison of Puro-PLA and single-antibody PLA for bassoon) - Supplemental Material
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[img] Image (JPEG) (Supplementary Figure 6: Incubation scheme and controls for GluA1 FUNCAT-PLA) - Supplemental Material
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[img] Image (JPEG) (Supplementary Figure 7: Influence of methionine starvation on Puro-PLA labeling) - Supplemental Material
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Abstract

Protein synthesis is a dynamic process that tunes the cellular proteome in response to internal and external demands. Metabolic labeling approaches identify the general proteomic response but cannot visualize specific newly synthesized proteins within cells. Here we describe a technique that couples noncanonical amino acid tagging or puromycylation with the proximity ligation assay to visualize specific newly synthesized proteins and monitor their origin, redistribution and turnover in situ.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1038/nmeth.3319DOIArticle
https://rdcu.be/cXQjPublisherFree ReadCube access
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4414919PubMed CentralArticle
ORCID:
AuthorORCID
Tirrell, David A.0000-0003-3175-4596
Schuman, Erin M.0000-0002-7053-1005
Additional Information:© 2015 Macmillan Publishers Limited. Received 28 April 2014; Accepted 29 January 2015; Published online 16 March 2015. We thank N. Fuerst and A. Staab for the preparation of cultured hippocampal neurons. E.M.S. is funded by the Max Planck Society; an Advanced Investigator award from the European Research Council, Deutsche Forschungsgemeinschaft (DFG) Collaborative Research Center (CRC) 902: Molecular Principles of RNA-based Regulation; DFG CRC 1080: Molecular and Cellular Mechanisms of Neural Homeostasis; and the DFG Cluster of Excellence for Macromolecular Complexes, Goethe University, Frankfurt. D.A.T is funded by US National Institutes of Health grant R01 GM062523.
Funders:
Funding AgencyGrant Number
Max Planck SocietyUNSPECIFIED
European Research Council (ERC)UNSPECIFIED
Deutsche Forschungsgemeinschaft (DFG)CRC 902: Molecular Principles of RNA-based Regulation
Deutsche Forschungsgemeinschaft (DFG)CRC 1080: Molecular and Cellular Mechanisms of Neural Homeostasis
NIHR01 GM062523
Issue or Number:5
PubMed Central ID:PMC4414919
Record Number:CaltechAUTHORS:20150205-174152503
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20150205-174152503
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:54465
Collection:CaltechAUTHORS
Deposited By: George Porter
Deposited On:17 Mar 2015 18:52
Last Modified:21 Apr 2020 17:51

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