LMB relocates NPM mutants in the nucleoplasm. (A) Confocal microscopy of NIH-3T3 cells transfected with pEGFP-C1-NPMmA or pEGFP-C1-NPMwt in presence and absence of LMB. In basal conditions, the mutant A is cytoplasmic (top left) but relocates in the nucleoplasm (top right) following incubation with LMB. eGFP-NPMwt localizes in the nucleoli both in the presence and absence of LMB (middle panel, left and right). The bottom panel shows LMB-treated cells transfected with pEGFP-C1-NPMmA (green) and immunostained with an antinucleolin (C23) monoclonal antibody (Texas red)–labeling nucleoli (arrow). The nuclear membrane is blue (TO-PRO-3 staining). After LMB, the eGFP-NPM mutant A was relocated in the nucleoplasm. Nucleolar exclusion was confirmed by an electronic cut of the nucleolar surface (inset in the bottom right figure). (B) The anti-NPM antibody 376 labels both nucleoli and cytoplasm of OCI/AML3 cells in the absence of LMB (left). Incubation with LMB results in nucleoplasmic relocation of the cytoplasmic NPM (right). Images were collected as described²⁷  using a Zeiss Plan Apochromat 100 ×/1.4 NA oil objective. (C) OCI-AML3 and U937 cell lysates were subjected to immunoprecipitation (IP) with either control IgG or rabbit anti-NPMm (Sil-C) and mouse anti-NPM (Cl. 376): Western blots with anti-Crm1 antibodies are shown in the top panels. Blotting with the anti-NPMm (Sil-C) and anti-NPM (Cl. 376) antibody are also shown (middle and bottom panels). WCL indicates OCI-AML3 whole-cell lysate included as control.