Approaches to Study Phosphorylation of Intermediate Filament Proteins Using Site-Specific and Phosphorylation State-Specific Antibodies

Abstract

This chapter discusses the production of site-specific and phosphorylation state-specific antibodies and a methodological approach to visualize the phosphorylation of intermediate filament (IF) proteins. It also presents a method to identify the phosphorylation sites of IF protein by protein kinase and to visualize the spatiotemporal phosphorylation by developing site-specific and phosphorylation state-specific antibodies. The rate of IF phosphorylation is affected by the concentration of IF protein and Mg2+, ionic strength, and temperature. The phosphorylation of IF proteins plays an important role in the dynamic IF reorganization. The development of site-specific and phosphorylation state-specific antibodies of IF proteins enables to monitor the spatiotemporal phosphorylation that occurs during various processes such as cell signaling and cell cycle. These site-specific and phosphorylation state-specific antibodies are available tools to analyze site-specific IF phosphorylation in vivo. The antibodies described in the chapter are also expected to have a wide application for the analysis of the phosphorylation of various proteins during cellular events.