Abstract
Present screening methods for protein-protein interactions (PPIs) rely on the overexpression of artificial fusion proteins, making it difficult to assess in vivo relevance. Here we combine stable isotope labeling with amino acids in cell culture (SILAC), RNA interference (RNAi), coimmunoprecipitation and quantitative mass-spectrometry analysis to detect cellular interaction partners of endogenous proteins in mammalian cells with very high confidence. We used this screen to identify interaction partners of β-catenin and Cbl.
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Acknowledgements
We thank B. Blagoev from the Center of Experimental Bioinformatics for fruitful discussions, J.V. Olsen and other members of our department for discussion and assistance, H. Clevers for generously providing us with the inducible β-catenin knockdown cell line and the Interaction Proteome project of the European Union for funding.
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Supplementary information
Supplementary Table 1
Sequences and normalized abundance ratios of the quantified peptides for β-catenin and three detected interaction partners. (DOC 38 kb)
Supplementary Table 2
Sequences and normalized abundance ratios of the quantified peptides for Cbl and four detected interaction partners. (DOC 39 kb)
Supplementary Table 3
Additional proteins identified with increased abundance ratios in the Cbl pulldown. (DOC 40 kb)
Supplementary Table 4
Ratio of proteins identified in the whole cell lysates after Cbl knock-down. (DOC 201 kb)
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Selbach, M., Mann, M. Protein interaction screening by quantitative immunoprecipitation combined with knockdown (QUICK). Nat Methods 3, 981–983 (2006). https://doi.org/10.1038/nmeth972
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DOI: https://doi.org/10.1038/nmeth972
