Nups were quantified in nuclear or NE extracts using targeted proteomics in combination with the spike-in of isotopically labeled peptides used as internal standard. For absolute quantification, the abundance of each Nup was calculated from the median ratio between the intensities of its endogenous proteotypic peptides (PTPs, light) and the corresponding spiked-in AQUA peptides (heavy). A panel of 90 MRM assays for 76 PTPs was used for absolute quantification. For relative quantification of Nup abundances across cell lines, crude synthetic peptides were used as internal standard instead of AQUA peptides. Therefore, a larger panel of 142 MRM assays for 119 PTPs was used. A detailed description of MRM assays development, data acquisition and processing is available in Supplementary Materials and Methods and Supplementary Figures S1E–G. A list of the MRM assays employed is available in Supplementary Table S1. Raw MRM data and method files are available at http://www.peptideatlas.org/PASS/PASS00189 for absolute quantification, and http://www.peptideatlas.org/PASS/PASS00188 for relative quantification across cell lines.