Investigation of Novel Cavin-1/Suppressor of Cytokine Signaling 3 (SOCS3) Interactions by Coimmunoprecipitation, Peptide Pull-Down, and Peptide Array Overlay Approaches.

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.