Nuclear export of NPM mutants is NES dependent. (A-B) H1299 cells transfected with cDNA encoding for the NPMwt and the NPM mutant proteins A to D⁸  in the absence (A) and presence (B) of LMB. eGFP-NPMwt localizes in the nucleoli both in the presence and absence of LMB. In the absence of LMB, all GFP mutants (A-D) show the expected aberrant cytoplasmic location (A). The same eGFP-mutants are completely relocated in the nucleus in the presence of LMB (B). (C-E) Time-course analysis of LMB induced nuclear retention of eGFP-NPMmutA in NIH-3T3 cells. (C) Confocal microscope images of NIH-3T3 cells at the indicated time points. ○ represents the regions (regions of interest [ROIs]) where the mean fluorescence emission of eGFP-NPMmutA was calculated in the experiment (eg, nucleus [N], cytoplasm [Cy], and the Golgi apparatus [G]). (D) Time-course measurements of mean fluorescence in the selected areas (ROIs) of NIH-3T3 cells. LMB addition produced a loss of fluorescence in the cytoplasm and Golgi apparatus and a concomitant increase in the mean fluorescence of the nucleoplasm. ImageJ software was used for the generation of ROIs and time-course analysis. (E) Western blot analysis of eGFP-NPMmutA subcellular distribution in NIH-3T3 cells with anti-GFP monoclonal antibody. LMB treatment induces a time-dependent accumulation of eGFP-NPMmutA in the pellet fractions (P). Purity of the subcellular fractions was assessed by stripping the membrane and reblotting with an anti–β-tubulin antibody (bottom panel). Only a nonsignificant contamination was detected for the overexpressed proteins (as evident in the GFP blotting) (middle panel). In untreated cells, eGFP-NPMmutA (except for the contaminant part) was found only in the cytoplasmic fractions (Cy). The experiment was conducted in absence of cycloheximide so that continuous presence of GFP-NPMmA in the cytoplasmic fraction during LMB treatment was detected with time.