Effect of RK424 on nuclear export of NP and the binding of NP to CRM1.

<p>(A) <i>In vitro</i> nuclear export assay incorporating NP-mRFP-Flag. Cells expressing NP-mRFP-Flag were permeabilized with digitonin and incubated for 1 h without (Buffer) or with HeLa cell lysate (+Lys) in the presence of the RK424 at the indicated concentrations (upper panels). Leptomycin B (LMB) was used as a positive control (LMB inhibits nuclear export of NP). Nuclei were stained with Hoechst 33342 (lower panels). The export of NP-mRFP-Flag was monitored by measuring mRFP fluorescence in the nucleus. Three independent experiments were performed and one representative result is shown. (B) Quantification of mRFP fluorescence in the nucleus. The nuclear localization of NP-mRFP-Flag was determined by monitoring mRFP fluorescence in the nucleus (> 100 cells). Data are expressed as the mean ± SD of three independent experiments. *;p<0.05 and **;p<0.005. (C) RK424 inhibits the binding of NP-mRFP-Flag to CRM1. FLAG agarose beads coupled to NP-Flag (NP-flag beads) were subjected to SDS-PAGE and the purity of the NP-Flag protein was checked by Coomassie Brilliant Blue (CBB) staining (left panel). HeLa cell lysate was incubated with NP-Flag beads in the absence or presence of RK424 (0.5, 5, or 10 μM). LMB was used as a positive control. Binding of CRM1 to NP was detected by western blotting with an anti-CRM1 MAb (right panel). EtOH and DMSO were used as vehicle controls. The positions of NP-Flag and CRM1 are indicated. Two independent experiments were performed and one representative result is shown.