Background

Insulin, secreted by the pancreatic β-cells, is the most powerful anabolic hormone known to regulate the metabolism of glucose, lipids, and amino acid metabolism through the activation of the insulin signaling pathway. Insulin binding to the insulin receptor (IR), a tetrameric complex consisting of two extracellular α-subunits and two transmembrane β-subunits, leads to a conformational change, the activation of tyrosine kinase activity in the β-subunits, and the transphosphorylation of β-subunits at Y972 (Sweet et al., 1987; Cheatham and Kahn, 1995; Yip and Ottensmeyer, 2003). The phosphorylation of the β-subunit at Y972 generates a NPXpY motif, which the downstream mediator insulin receptor substrate (IRS) subsequently recognizes and binds to in the insulin receptor b (IRβ), resulting in the activation of PI3K-AKT signaling (Machado-Neto et al., 2018; White et al., 1988). Therefore, the binding of the IRS to the IRβ subunit plays a critical role in the activation of insulin signaling (Peng and He, 2018).

Far-western blotting is an effective technique to assess protein–protein interactions, including receptor–ligand interactions (Kaido et al., 2007; Wu et al., 2007), in in vitro assays. In a far-western blotting analysis, one protein is first separated in an SDS-PAGE gel and transferred to a membrane, followed by the binding of a non-antibody secondary protein. Then, a specific antibody against the secondary protein will be employed to determine its binding to the first protein that is being transferred onto the membrane (Figure 1). This method can be used in a regular laboratory, without the need of expensive equipment, to determine the interaction of proteins in other methods, such as the surface plasmon resonance system. We introduce a far-western blotting analysis that has been successfully used to determine the interaction of IRβ to its downstream mediator, the IRS, and to assess the importance of post-translational acetylation of IRS in affecting its binding to IRβ, as well as the activation of the insulin signaling pathway in our studies (Cao et al., 2017; Peng et al., 2022).

Figure 1. Procedure of far-western blotting to examine the binding of IRS to IRβ. A. Sequential steps of the procedure. B. Diagram depicts the detection of the binding with antibody. C. Purified IRS1 and IRS2 proteins were subjected to SDS-PAGE and stained with colloidal blue. D. Similar quantities of IRS2 and acetylated IRS2 by acetyltransferase P300 protein were employed in an SDS-PAGE and transferred onto a membrane; after renaturation, membranes were incubated with IRβ, followed by incubation with anti-IRβ antibody.