Figure 6.

Inhibition of NF-κB/IκBα nucleocytoplasmic shuttling by a p50 NLS peptide and a LMB-insensitive CRM1 mutant. (A) Empty vector, CRM1 wild type (WT), or LMB-insensitive Crm1-K1 mutant was transfected into HEK293 cells stably expressing GFP-IκBα. Cells were then treated or untreated with LMB (20 ng/ml for 15 min) and visualized with GFP fluorescence. (B) HeLa cells were pretreated with either LMB (20 ng/ml for 30 min; lane 4) or cell-permeable p50 NLS peptide (NF-κB SN50) for 1 h (100 μg/ml; lane 3) and stimulated with TNFα (10 ng/ml for 15 min; lanes 2 and 3). Nuclear extracts were analyzed for NF-κB activity with EMSA (Upper). Cytoplasmic extracts from the same samples were analyzed by Western blotting for IκBα and α-tubulin (loading control). (C) HeLa cells were untreated or treated with SN50 (100 μg/ml for 1 h) and then either untreated (Top) or treated with LMB (20 ng/ml for 30 min; Middle) and TNFα (10 ng/ml for 30 min; Lower). Cells were then stained with p65 antibody as described above. (D) Model of nucleocytoplasmic shuttling of the inactive NF-κB/IκBα complexes. See the text for details.