Localization of Xpo-t and XpN in semipermeabilized HeLa cells. The Ran system promotes nuclear accumulation. (A) Xpo-t labeled with Alexa-546 (0.5 μM) and an energy regeneration system was combined with buffer (control) or the indicated reagents and then incubated with digitonin-permeabilized HeLa cells for 10 min at room temperature, fixed with paraformaldehyde, and spun onto coverslips, and images were collected by confocal microscropy. The Ran system (NRGB) contains 0.4 μM NTF2 (N), 4 μM RanGDP (R), 0.4 μM RanGAP (G), and 0.4 μM RanBP1 (B). The ΔN44 protein is composed of amino acids 45 to 465 of importin β and was added at 1 μM. (B and C) Localization of XpN-Alexa-546 (B) and XpN mut-Alexa-546 (F54A/F55A) (C) as in panel A.