FIG. 5.
Nuclear import of IκBα is energy and temperature dependent. Nuclear import reactions into digitonin-permeabilized HeLa cells were performed with either GST-IκBα (A to D) or GST-NLS (E to H). Leptomycin B (10 nM) was included in the nuclear import reactions with GST-IκBα (A to C). The import reactions were carried out under standard conditions (A and E), in the presence of WGA (B and F), on ice (C and G), or in the presence of 25 U of apyrase per ml (D and H). The cellular localization of the import substrates was determined by indirect immunofluorescence with a monoclonal antibody directed against GST. Panels A and E were from equivalent exposure settings. Panels B to D and F to H were exposed for 10 to 20 times longer than panels A and E.