We used a buffer containing 400 mM KCl, 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 5 mM ethylenediaminetetraacetic acid, 0.4% NP-40, protease inhibitors, and 5% glycerol to obtain cell lysates by sonication. We incubated the cell lysates with an anti-importin β antibody overnight at 4°C. Then, we used protein A/G PLUS-Agarose beads (sc-2003; Santa Cruz) to incubate the reaction mixture for 2 h at 4°C. The precipitates were then washed three times with a washing buffer and eluted from the beads by boiling with 1 × sodium dodecyl sulfate (SDS) for 5 min at 95°C. The protein samples were detected by SDS-polyacrylamide gel electrophoresis.

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