Heparin sepharose beads

The proteins were added to transport buffer (20 mM HEPES/NaOH, pH 7.4, 110 mM potassium acetate, 2 mM magnesium acetate, 5 mM sodium acetate, 0.5 mM EGTA/NaOH, 2 mM DTT) in the presence or absence of anti-importin α1 mAb, and mixed with glutathione-sepharose 4B beads (GSH-beads, GE Healthcare) or heparin-sepharose beads (Sigma). The mixtures were incubated at 4 °C for 1 h and then washed with transport buffer. Bound proteins were eluted with sample buffer for sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Supernatants from Hi-5 adherent cells containing recombinant proteins were incubated with heparin-sepharose beads (Sigma-Aldrich, Germany) for 1.5 h at 4°C. Recombinant proteins were detected with the anti His-tag antibody by Western blotting prior to the pulldown to ensure that similar amounts of all recombinant proteins were used in the assay. The binding of recombinant proteins to the heparin-beads was competed by adding 0.1 to 2 mg of soluble heparin (Sigma-Aldrich, Germany) to the binding reaction. As negative control, agarose beads lacking heparin were used (Sigma-Aldrich, Germany). The beads were washed three times with PBS and the proteins were eluted with denaturalizing loading buffer for SDS-PAGE. The presence of the recombinant proteins was detected by Western blotting using the anti His-tag antibody.