NES and tryptophan(s) mutations act in concert to export NPM mutants. Confocal microscope analysis of NIH-3T3 transfected with either pEGFP-C1-NPMwt or different pEGFP-C1-NPM mutants. NPM mutant A localizes exclusively in the cytoplasm. The NPM mutant E, retaining tryptophan 288 but lacking tryptophan 290, partially relocalizes in the nucleoli when the nuclear export of the protein is blocked by LMB treatment. Reinsertion of the tryptophan 290 (NPMmutA, A290W) produced an almost identical effect. When both tryptophans (288 and 290) were reinserted in the mutant A, the eGFP-tagged protein totally localized into the nucleoli, despite the 2 NES motifs, whether LMB was present or not. Images were collected as described²⁷  using a Zeiss Plan Apochromat 100 ×/1.4 NA oil objective.