In vitro Nuclear Export Assay

We performed in vitro nuclear export experiment with slight modifications (Cassany and Gerace, 2009 (link)). First, 1 µM GST-NES^PKI-NLS^IBB (GST-NES-NLS), 1 µM KPNB1, 1 µM NTF2, 1 × energy regeneration system (Cassany and Gerace, 2009 (link)), 0.01% Triton-X100, and 2 µM of hRan were added to semi-permeabilized HeLa cells and incubated at room temperature for 60 mins to accumulate nuclear cargoes. The very low concentration of Triton-X prevents non-specific cytoplasmic binding, but does not permeate nuclear envelopes (Figure 3—figure supplement 1). After washing, the cells were incubated with either human or yeast proteins (0.1 µM CRM1, 1 µM Ran and 1 µM RanBP1), energy regeneration system, 0.01% Triton-X, for different time points at room temperature with gentle shaking. After reaction, the cells were washed, fixed and visualized by immunostaining with GST antibody (Santa Cruz, 1:400). Statistics were based on measurements from at least 30 cells for each sample, and statistical significance was calculated by one-way ANOVA test in Graphpad software.