SEC–MALS analysis of purified Nup358-KLC2/BicD2-CTD complexes. The Nup358-KLC2-fusion protein (WT or W2224A/D2225A mutant) was mixed with the BicD2-CTD in a 1:1 molar ratio, and the complex was isolated by size exclusion chromatography to remove unbound reactants. These complexes were subsequently analyzed by SEC–MALS. The Rayleigh ratio (distinct colors) and molar mass MW (colored in shades of red, orange, and pink) are plotted vs elution volume (see also Table S3). The SDS–PAGE of the elution fractions is shown beneath the SEC–MALS profiles of the complexes. (A) The Nup358-KLC2-fusion protein and the purified Nup358-KLC2-fusion protein/BicD2-CTD complex (5 mg/mL) were analyzed. (B) Same experiment as in panel A, at 2.5 mg/mL. (C) Same experiment as in panel A, with the Nup358/W2224A/D2225A-KLC2-fusion protein. Numbers of replicates for each experiment: (A) 2 and 3, (B) 4 and 2, and (C) 3 and 3 (see Table S3).