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Pairwise detection of site-specific receptor phosphorylations using single-molecule blotting

Title 
Pairwise detection of site-specific receptor phosphorylations using single-molecule blotting
Authors 
Kim, K.L., Kim, D., Lee, S., Kim, S.-J., Noh, J.E., Kim, J.-H., Chae, Y.C., Lee, J.-B., Ryu, S.H.; 이종봉
Issue Date 
2016-03
Journal Title 
Nature communications
Volume 
7
Issue 
11107
Publisher 
Nature publishing group
Abstract 
Post-translational modifications (PTMs) of receptor tyrosine kinases (RTKs) at the plasma membrane (PM) determine the signal transduction efficacy alone and in combination. However, current approaches to identify PTMs provide ensemble results, inherently overlooking combinatorial PTMs in a single polypeptide molecule. Here, we describe a single-molecule blotting (SiMBlot) assay that combines biotinylation of cell surface receptors with single-molecule fluorescence microscopy. This method enables quantitative measurement of the phosphorylation status of individual membrane receptor molecules and colocalization analysis of multiple immunofluorescence signals to directly visualize pairwise site-specific phosphorylation patterns at the single-molecule level. Strikingly, application of SiMBlot to study ligand-dependent epidermal growth factor receptor (EGFR) phosphorylation, which is widely thought to be multi-phosphorylated, reveals that EGFR on cell membranes is hardly multi-phosphorylated, unlike in vitro autophosphorylated EGFR. Therefore, we expect SiMBlot to aid understanding of vast combinatorial PTM patterns, which are concealed in ensemble methods, and to broaden knowledge of RTK signaling.
Keywords 
POSTTRANSLATIONAL MODIFICATION CODES, PROTEIN MODIFICATIONS, PULL-DOWN, PROTEOMICS, RECONSTRUCTION, ACTIVATION, MICROSCOPY, MEMBRANE
URI 
http://oasis.postech.ac.kr/handle/2014.oak/29995
DOI 
http://dx.doi.org/10.1038/NCOMMS11107
ISSN 
2041-1723
Article Type 
ARTICLE
Appears in Collections
Physics (물리학과) > 1. Journal Papers

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