Phos-tag Immunoblot Analysis for Detecting IRF5 Phosphorylation

Go R Sato; Tatsuma Ban; Tomohiko Tamura

doi:10.21769/BioProtoc.2295

Phos-tag Immunoblot Analysis for Detecting IRF5 Phosphorylation

Bio Protoc. 2017 May 20;7(10):e2295. doi: 10.21769/BioProtoc.2295.

Authors

Go R Sato¹, Tatsuma Ban¹, Tomohiko Tamura¹

Affiliation

¹ Department of Immunology, Yokohama City University Graduate School of Medicine, Yokohama, Japan.

Abstract

While the activation of the transcription factor interferon regulatory factor 5 (IRF5) is critical for the induction of innate immune responses, it also contributes to the pathogenesis of the autoimmune disease systemic lupus erythematosus (SLE). IRF5 phosphorylation is a hallmark of its activation in the Toll-like receptor (TLR) pathway, where active IRF5 induces type I interferon and proinflammatory cytokine genes. By using the phosphate-binding molecule Phos-tag, without either radioisotopes or phospho-specific antibodies, the protocol described here enables detection of the phosphorylation of both human and murine IRF5, as well as that of other proteins.

Keywords: IRF5; Immunoblot; Innate immunity; Phos-tag; Phosphorylation; SDS-PAGE; SLE; TLR.