Abstract
The cyclic GMP-AMP synthase (cGAS) is the principal DNA sensor, which binds DNA and triggers the type I interferon production. We used ISD45 or inactivated Vaccinia Virus (VACV) to stimulate cGAS and monitored cellular localization by immunofluorescence microscopy, Operetta high-content screening, and cytoplasmic/nuclear fractionation. LocNES server was used to predict cGAS nuclear export signal (NES) sequence and characterized the function by mutagenesis. This protocol provides a prototype of cGAS subcellular distribution or the identification of NES in other proteins. For complete details on the use and execution of this protocol, please refer to Sun et al. Sun et al. (2021).