NES-dependent altered nucleocytoplasmic traffic of NPM in NPMC⁺ AML. (A) Subcellular distribution of wild-type and mutant NPM in NPMc⁺ AML cells. Data of experiments on 2 NPMc⁺ AML patients are shown. The anti-NPMm blotting (both patients, top panels) shows accumulation, upon LMB treatment, of NPM mutant protein in the insoluble fraction (I) (both patients, lane 9, top panels). Relocation, upon LMB treatment, of NPM wild-type protein in the same fraction was demonstrated by blotting the membrane with an anti-NPMwt–specific antibody (both patients, lane 9, middle panels). The insoluble fraction represents mainly subcellular fraction containing most of the nucleolar compartment, as assessed by stripping the membrane and reblotting for the nucleolar marker fibrillarin (both patients, bottom panels, lanes 3, 6, and 9), and possibly other insoluble nuclear structures. NPMm indicates mutated NPM; NPMwt, wild-type NPM. (B) Interaction between wild-type NPM, leukemic NPM mutants, and Crm1 in NPMc⁺ AML cells. Cell lysates from either NPMc^– or NPMc⁺ AML patients were subjected to immunoprecipitation (IP) with either control IgG (not shown), mouse monoclonal anti-NPMwt antibody from Invitrogen (Pts 4, 2, and 3), or rabbit polyclonal antibody anti–mutated NPM (anti-NPMm, Sil-C) (Pts 2 and 3), as indicated. Coprecipitation of either NPM mutant protein or wild-type NPM, in the reciprocal experiments, was detected by Western blot with the anti-NPMm–specific antibody Sil-C (middle panels) and anti-NPMwt–specific antibody (bottom panels), respectively. Stechiometry of the NPMwt/NPMm protein complex changes with patients. Western blot analysis with a rabbit polyclonal antibody anti-Crm1 shows a much stronger signal in the NPMc⁺ than NPMc^– AML sample (top panels). OCI/AML3 cell lysate was included as control for antibody activity. NPMm indicates mutated NPM; NPMwt, wild-type NPM.