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Nuclear translocation of proteins is an essential aspect of normal cell function, and defects in this process have been detected in many disease‐associated conditions. The detection and quantification of nuclear translocation was significantly boosted by the association of robotized microscopy with automated image analysis, a technology designated as high‐content screening. Image‐based high‐content screening and analysis provides the means to systematically observe cellular translocation events in time and space in response to chemical or genetic perturbation at large scale. This approach yields powerful insights into the regulation of complex signaling networks independently of preconceived notions of mechanistic relationships. In this review, we briefly overview the different mechanisms involved in nucleocytoplasmic protein trafficking. In addition, we discuss high‐content approaches used to interrogate the mechanistic and spatiotemporal dynamics of cellular signaling events using Forkhead box O (FOXO) proteins and the nuclear factor‐κB (NF‐κB) as important and clinically relevant examples....
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