Mutations in NFE2 alter transactivating activity and subcellular localization. (A) Electrophoretic mobility shift assay (EMSA) of wt-NFE2 and NFE2 mutants. Nuclear extracts of HEK-293 cells transfected with expression vectors encoding either wt-NFE2 (lanes 1-3), or the indicated mutants (lanes 4-10), together with MafG, were incubated with a ³²P-labeled oligonucleotide containing a NFE2 binding site, as previously described.¹⁰  In lane 2, a 100× excess of a nonradioactive oligonucleotide, encoding the consensus NFE2 binding site, was added. Open circle: unspecific band; triangle: NFE2 band. (B) CB3 cells, which lack endogenous NFE2, were transduced with wt-NFE2 or various NFE2 mutants, as indicated, and assayed for β-globin and β-2-microglobulin housekeeping gene expression by quantitative reverse transcription (qRT)-PCR, as previously described.⁹  Results represent the mean ± SEM of at least 4 independent experiments and are reported as relative expression levels setting β-globin expression in empty transfected CB3 cells at 1. Data were analyzed for statistical significance by 1-way ANOVA with Bonferroni’s post hoc multiple comparison test. *P < .05; **P < .01; ***P < .001. (C) CB3 cells were transduced with pLeGO-iC2-NFE2 as well as with either an empty pLeGO-iG vector, or with pLeGO-iG vectors encoding either wt-NFE2 or the NFE2 mutants indicated. Cells were assayed for β-globin and β-2-microglobulin housekeeping gene expression by qRT-PCR, as in panel B. Results represent the mean ± SEM of at least 4 independent experiments and are reported as relative expression levels setting β-globin expression in pLeGO-iC2-NFE2/empty pLeGO-iG-transduced CB3 cells at 1. Data were analyzed for statistical significance by 1-way ANOVA with Bonferroni’s post hoc multiple comparison test. *P < .05; **P < .01; ***P < .001; **** P < .0001. (D, top) Schematic representing the NFE2 protein indicating functional domains. (Bottom) A table summarizing the data from panels A-C and E. c, cytoplasmic; n, nuclear; n.d., not determined; *Published NFE2 mutants.¹⁰  **The R273W mutant weakly binds DNA in an EMSA (Type IIa-like), but not in a ChIP assay (type IIb-like). Protein nomenclature, see supplemental Table 2. (E-F) Subcellular localization. 293T cells were transduced with pLeGO-iG expressing the NFE2 mutants indicated: Cells were fixed, permeabilized, and stained with an antibody against NFE2, detected by a secondary antibody coupled to AF647 (red stain), counterstained with 4′,6-diamidino-2-phenylindole (DAPI, blue) and assessed by immunofluorescence microscopy. (G, left) CB3 cells were transduced with an empty vector, wt-NFE2 or the R237W-NLS mutant indicated and assayed by qRT-PCR as cells in panel B). n = 4 per construct. (Right) CB3 cells were transduced with pLeGO-iC2-NFE2, as well as with either an empty pLeGO-iG vector or with pLeGO-iG vectors encoding either wt-NFE2 or the R273W-NLS mutant. Cells were assayed by qRT-PCR as in panel B. n = 3 per construct. WT, wild-type.