Isolation of Redox-Active Endosomes (Redoxosomes) and Assessment of NOX Activity

Abstract

Reactive oxygen species (ROS) convey signals essential for proliferation, maintenance, and senescence of a growing list of cell types. Compartmentalization of these signals is integral to cell viability as well as the signaling pathways ROS direct. Redox-active endosomes (redoxosomes) are formed downstream of several ligand-activated receptors. NADPH oxidase (NOX) is a main component of redoxosomes, which recruits multiple proteins (Rac1, NOX2, p67^phox, SOD1). Isolation of redoxosomes and evaluation of how superoxide (O₂˙^-) production directs receptor signaling at the level of the endosome have enabled a better understanding of biologic processes controlled by ROS. In this chapter, we will first review the major signaling pathways that utilize redoxosomes and components that control its redox-dependent functions. We will then outline biochemical and biophysical methods for the isolation and characterization of redoxosome properties.

Keywords: EPR; Immuno-affinity isolation; Iodixanol; Lucigenin; NOX; ROS; Rac1; Redoxosomes; TRAF.