Purification and complex formation of NTRs

For complex formation, His-NOSIP and purified NTRs were incubated in TPB at a molar ratio of 1:1 (except His-importin β:His-importin 7:His-NOSIP and His-importin α:His-importin β:NOSIP with a ratio of 1:1:3) in the presence or absence of a 3-fold molar excess of RanQ69L₁₋₁₈₀-GTP for 1 to 2 h at 4 °C. Samples were cleared by centrifugation at 4 °C at 16,100g for 20 min and subjected to size exclusion chromatography using a Superdex S200 analytical increase 10/300 GL column (Cytiva) equilibrated in TPB. Fractions were analyzed by SDS-PAGE followed by Coomassie staining.