Binding of NP to importin α.

<p>(A) Purification of mRFP-Flag-tagged WT and mutant NP110aas from COS-7 cells and GST-tagged importin α isoforms, Rch1, Qip1 and NPI-1 from <i>Escherichia coli</i>. For purification of NP110aa, COS-7 cells were transfected with pCAGGS encoding mRFP-Flag-tagged WT and mutant NP110aas, lysed and proteins were purified using ANTI-FLAG M2 Agarose beads and the 3× Flag peptide. For purification of importin α proteins, Escherichia coli expressing GST-tagged Rch1, Qip1 and NPI-1 were lysed and proteins were purified using glutathione-Sepharose 4B. Purified proteins were subjected to 10% or 15% SDS-PAGE and stained by Coomassie brilliant blue. (B) Binding of WT and mutant NP110aas to the three importin α isoforms. Both GST beads bound to Rch1, Qip1, NPI-1 and non-bound GST beads were incubated with the WT, 14-110aa, R8A or S9A NP110aa proteins. Binding of NP110aa to importin α was detected by Western blotting with anti-FLAG M2 monoclonal antibody (mAb). (C) Quantification of band intensity. Different concentrations of Rch1, Qip1 and NPI-1 proteins (0, 5, 25 and 50 µM) were subjected to 10% SDS-PAGE and Western blotting with anti-Rch1, -Qip1 and -NPI-1 mAbs. Band intensities were measured by Image J and the standard curves for the amount of importin α isoforms were constructed. (D) Competition binding assay of importin α to WT and S9A NP110aa-binding beads. Purified importin α isoforms, Rch1, Qip1 and NPI-1 from <i>Escherichia coli</i> were subjected to 10% SDS-PAGE and stained with Coomassie brilliant blue. All three importin α proteins (50 µM) were incubated with WT and S9A NP110aa-binding Flag beads at 4°C for 1 day. After washing with buffer, the binding of importin α to WT and S9A NP110aa was detected by Western blotting with anti-Rch1, -Qip1 and -NPI-1 mAbs. Empty Flag beads were used as the negative control. The amount of importin α proteins which bound to WT NP110aa and S9A NP110aa were calculated by analyzing the standard curves as shown in C right panel. (E) Binding of WT full length NP, WT and S9A NP110aa to the three importin α isoforms in HEK-293T cells. HEK-293T cells were transfected with pCAGGS encoding mRFP-Flag-tagged WT full length NP, WT or S9A NP110aa, cells were lysed and proteins were purified using ANTI-FLAG M2 Agarose beads and the 3× Flag peptide. The bound importin αs to NP was detected by Western blotting with anti-Rch1, -Qip1 and -NPI-1 mAbs. The same amount of lysate was used for control. The amounts of importin α proteins which bound to WT NP, WT NP110aa and S9A NP110aa were calculated by analyzing the standard curves as shown in C right panel.