293T cells were transfected with an expression vector containing the FLAG-tagged protein of interest. Cells were harvested and lysed with Triton X-100 buffer [25 mM Hepes (pH7.5), 100 mM NaCl, 10% glycerol, 1 mM EDTA, and 1% Triton X-100] supplemented with protease inhibitor cocktail (Roche). WCLs were sonicated and centrifuged at 12,000 rpm for 30 min. Supernatant was filtered, precleared with protein A/G agarose beads at 4°C for 1 hour, and then incubated with anti-FLAG M2 agarose beads at 4°C for 4 hours. Agarose beads were washed extensively with lysis buffer and eluted with 3×FLAG peptide (0.2 mg/ml). Eluted proteins were analyzed by SDS—polyacrylamide gel electrophoresis (PAGE) and Coomassie/silver staining.

For recombinant GST-RTA(N) and GST–importin β purification, E. coli BL21 (DE3) was induced with 0.1 mM isopropyl-β-d-thiogalactopyranoside at 20°C overnight. Cell pellets were harvested and lysed. After sonication and centrifugation, cell lysates were incubated with glutathione Sepharose 4B beads (GE Healthcare) for 4 hours at 4°C. Sepharose beads were washed extensively with lysis buffer, and GST-RTA(N) was eluted with 10 mM reduced glutathione. Purified GST, GST-RTA(N), and GST–importin β were analyzed by SDS-PAGE and Coomassie staining.

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