A leucine‐rich nuclear export signal in the p53 tetramerization domain: regulation of subcellular localization and p53 activity by NES masking The intrinsic.

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A leucine‐rich nuclear export signal in the p53 tetramerization domain: regulation of subcellular localization and p53 activity by NES masking The intrinsic p53 NES is sufficient to mediate nuclear export. (A) Subcellular localization of p53 without MDM2. Plasmid expression constructs encoding 22/23–GFP (MDM2‐binding deficient), wild‐type p53–GFP or L348,350A–GFP were electroporated into Saos‐2 or p53−/−/mdm2−/− mouse embryonic fibroblasts (MEF) and fixed and stained with Texas Red–phalloidin to visualize cytoplasmic actin. Images of representative cell fields were captured on an epifluorescence microscope. More than 1000 cells were analyzed for each construct. (B) Shuttling of a p53 mutant which does not bind MDM2. Saos‐2 cells (human) were electroporated with 22/23–GFP, pre‐treated with cycloheximide alone or with LMB and cycloheximide, and then fused to non‐expressing Balb/c 3T3 cells (mouse) in the presence of cycloheximide. Fused cells were incubated and fixed as described in Figure 1C. Each image is representative of >50 fusions on each coverslip. (C) Shuttling of a heterologous protein mediated by the p53 NES. A wild‐type peptide consisting of amino acids 339–352 was covalently conjugated to FITC‐labeled BSA. wt NES–BSA–FITC was then microinjected along with 70 kDa rhodamine–dextran (an inert marker which enables localization of the injection site) into the nuclei of untreated or LMB pre‐treated HeLa cells. The injected cells were incubated for 4 h with or without the drug at 37°C to allow sufficient time for export. Images were captured on a confocal microscope. IF THIS IMAGE HAS BEEN PROVIDED BY OR IS OWNED BY A THIRD PARTY, AS INDICATED IN THE CAPTION LINE, THEN FURTHER PERMISSION MAY BE NEEDED BEFORE ANY FURTHER USE. PLEASE CONTACT WILEY'S PERMISSIONS DEPARTMENT ON PERMISSIONS@WILEY.COM OR USE THE RIGHTSLINK SERVICE BY CLICKING ON THE 'REQUEST PERMISSIONS' LINK ACCOMPANYING THIS ARTICLE. WILEY OR AUTHOR OWNED IMAGES MAY BE USED FOR NON-COMMERCIAL PURPOSES, SUBJECT TO PROPER CITATION OF THE ARTICLE, AUTHOR, AND PUBLISHER. EMBO J, Volume: 18, Issue: 6, Pages: 1660-1672, First published: 15 March 1999, DOI: (10.1093/emboj/18.6.1660)