Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2013 May 6;210(5):1003–1019. doi: 10.1084/jem.20120521

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© 2013 Jutzi et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

PMC Copyright notice

Figure 2. — Transactivating activity of mutant NF-E2 proteins and their effect on WT NF-E2 activity. Plasmids encoding a β-globin promoter-luciferase construct (A; Igarashi et al., 1994) or a reporter construct encoding 5.2 kb of the β1-tubulin promoter coupled to the luciferase reporter gene (B) were cotransfected into HEK-293 cells either with expression vectors for MafG (white bars), WT NF-E2 (black bars), or various NF-E2 mutants (gray bars) as indicated. Luciferase activity was measured 16 h after transfection and normalized for transfection efficiency by determination of Renilla luciferase activity from a cotransfected vector. Activity of MafG alone was set at 1 and fold activity relative to this control is depicted. Bar graphs represent the mean ± SEM of four independent experiments, each performed in duplicate. Statistical analysis was done by one-way ANOVA with Bonferroni’s post-hoc multiple comparison test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (C) CB3 cells, which lack endogenous NF-E2, were transfected with WT NF-E2 or various NF-E2 mutants as indicated. 72 h after transfection, RNA was harvested and assayed for β-globin and β-2-microglobulin housekeeping gene expression by qRT-PCR. Results represent the mean ± SEM of four independent experiments and are reported as relative expression levels setting β-globin expression in WT NF-E2 transfected CB3 cells at 100. Data were analyzed for statistical significance by one-way ANOVA with Bonferroni’s post-hoc multiple comparison test. ***, P < 0.001. (D) Experimental design. CB3 cells were transduced with pLeGO-iG-NF-E2, sorted for GFP expression, and a single clone (CB3-NF-E2wt), which displays WT NF-E2 expression, was selected. Subsequently, CB3-NF-E2wt cells were transduced with either an empty pLeGO-iT2 vector or with pLeGO-iT2 vectors encoding the indicated NF-E2 mutants. (E) Double-positive cells were FACS sorted and assayed for β-globin and β-2-microglobulin housekeeping gene expression by qRT-PCR. Results represent the mean ± SEM of four independent experiments and are reported as relative expression levels setting β-globin expression in empty pLeGO-iT2 transduced CB3-NF-E2wt cells at 1. Data were analyzed for statistical significance by one-way ANOVA with Bonferroni’s post-hoc multiple comparison test. **, P < 0.01. (F) Peripheral blood of transplanted mice expressing the indicated NF-E2 mutants and of control mice was FACS sorted for CD71, Ter-119 double-positive cells. RNA from sorted cells was assayed for β-globin and β-2-microglobulin housekeeping gene expression by qRT-PCR. Results represent the mean ± SEM of four to five animals in each group and are reported as relative expression levels setting β-globin expression in a mouse transduced with empty pLeGO-iG at 1. Data were analyzed for statistical significance by Student’s t tests. *, P < 0.05; **, P < 0.01.