A Toolbox for Quantifying Nuclear and Nucleolar Protein Accumulation Using NLS and NoLS Fusion Reporters

Fluorescent proteins that are fused to nuclear localization signals (NLSs) or nucleolar localization signals (NoLSs) are commonly used to investigate the accumulation of proteins within the nucleus and nucleolus. These reporters allow for the in vivo evaluation of signal sequence function, independent of the surrounding protein context. Here, we present a standardized protocol for measuring nuclear and nucleolar accumulation using NLS- and NoLS-fusion reporters. The workflow integrates plasmid construction, transfection, expression control, imaging and computational analysis to minimize artifacts and enhance reproducibility. This framework provides a robust basis for the reliable quantification of NLS- and NoLS-mediated localization and allows for the comparison of nuclear transport mechanisms across studies.