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Investigation of novel cavin-1/suppressor of cytokine signaling 3 (SOCS3) interactions by coimmunoprecipitation, peptide pull-down, and peptide array overlay approaches

Williams, Jamie J. L.; Baillie, George S.; Palmer, Timothy M.

Authors

Jamie J. L. Williams

George S. Baillie

Timothy M. Palmer



Contributors

Cedric M. Blouin
Editor

Abstract

The ability of inducible regulator suppressor of cytokine signaling 3 (SOCS3) to inhibit Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling requires interaction with specific cytokine receptors, JAKs, and components of the cellular ubiquitylation machinery. However, it is now clear that additional protein interactions are essential for effective inhibition of JAK-STAT signaling that have also identified new roles for SOCS3. For example, we have demonstrated that SOCS3 interaction with cavin-1, a core component of caveolae essential for their formation, is required for effective inhibition of interleukin (IL)-6 signaling and maintenance of cellular levels of caveolae. This is achieved through cavin-1 interaction with a discrete motif within the SOCS3 SH2 domain. Here, we describe in detail three methods (coimmunoprecipitation; peptide pull-down; peptide array overlay) we have used to validate and characterize cavin-1/SOCS3 interactions in vitro.

Citation

Williams, J. J. L., Baillie, G. S., & Palmer, T. M. (2020). Investigation of novel cavin-1/suppressor of cytokine signaling 3 (SOCS3) interactions by coimmunoprecipitation, peptide pull-down, and peptide array overlay approaches. In C. M. Blouin (Ed.), Caveolae: methods and protocols (105-118). New York: Springer. https://doi.org/10.1007/978-1-0716-0732-9_10

Acceptance Date Apr 11, 2020
Online Publication Date Jun 17, 2020
Publication Date 2020
Deposit Date Jul 27, 2020
Publisher Springer
Pages 105-118
Series Title Methods in molecular biology
Series Number 2169
Book Title Caveolae: methods and protocols
ISBN 9781071607312
DOI https://doi.org/10.1007/978-1-0716-0732-9_10
Keywords Coimmunoprecipitation; Peptide array; Peptide pull-down; Protein-protein interactions; JAK-STAT; Caveolae; Cavin-1; SOCS3
Public URL https://hull-repository.worktribe.com/output/3548197
Additional Information First Online: 17 June 2020