Binding of NP-NES3 mutant proteins to CRM1.

Abstract

<p>(A) FLAG-tagged NP-NES3 wild-type and mutant (Φ1, Φ2, Φ3, and Φ4) proteins were prepared by transfecting the relevant pCAGGS plasmids into HEK-293T cells. The cells were then lysed. The proteins were captured by anti-FLAG agarose beads and eluted with a FLAG-peptide. CRM1-HA agarose beads were prepared by transfecting CRM1-HA/pCAGGS into HEK-293T cells. The cells were then lysed and the proteins were captured on anti-HA agarose beads. The purified proteins and beads were then run in 10% SDS-PAGE gels and stained with Coomassie Brilliant Blue. (B) NP/CRM1 binding was demonstrated by incubating equal amounts of CRM1-HA agarose beads or anti-HA agarose beads alone with purified NP proteins at 4°C for 3 h. After pull-down and washing, the beads were boiled with 4×SDS sample buffer and subjected to 10% SDS-PAGE and Western blot analysis with an anti-WSN Ab and an anti-CRM1 MAb. Input NP at 30% was included. (C) Equal amounts of purified wild-type NP-NES3-FLAG or mutant (Φ1, Φ2, Φ3, and Φ4) protein were co-incubated with CRM1-HA agarose beads at 4°C for 3 h, pulled-down, washed, boiled with 4×SDS sample buffer, and then subjected to Western blot analysis with an anti-WSN Ab and an anti-CRM1 MAb. NP/CRM1 binding affinity was compared by measuring the intensity of the NP band normalized against the CRM1 band. Each 30% input NP protein and remained NP protein after binding with the CRM1-HA agarose beads are also shown.</p