Fig. 1. Effect of CRM1 inhibition on STAT2 nucleocytoplasmic distribution in untreated and IFN-ß-stimulated 2fTGH cells. (A) 2fTGH cells were either not treated or were treated with IFN-ß for the indicated time periods in the absence (upper panel) or presence of LMB (10 ng/ml) (lower panel). The cells were fixed and stained for STAT2 by indirect immunofluorescence. Fluorescence micrographs were obtained by confocal laser-scanning microscopy. (B) 2fTGH cells were treated as described in A. In addition, LMB alone was added to the cells for 4 hours. Whole-cell extracts were resolved by SDS-PAGE and activated STAT2 was detected with a phosphospecific STAT2-Tyr689 antibody (top). Loading of equal amounts of STAT2 was confirmed by reprobing with STAT2 antiserum (bottom). (C) 2fTGH cells were either left untreated or were treated with LMB (10 ng/ml) for the indicated time periods. Cells were stained for STAT2 and indirect immunofluorescence was analyzed by confocal laser-scanning microscopy. Staining profiles (line-scan analysis) of STAT2 are shown in the lower panel. (D) U2A cells were either left untreated or treated with LMB (10 ng/ml) for the indicated time periods. Cells were stained for STAT2 and analyzed by confocal laser-scanning microscopy. Staining profiles of STAT2 are shown in the lower panel. (E) U2A cells were transiently transfected with STAT2-EGFP expression plasmid. Fourty hours after transfection, subcellular localization of STAT2-EGFP was determined by fluorescence microscopy before and after 2 and 4 hours of LMB treatment.