Figure 3.
Figure 3. Forkhead responsive elements present in the gilz promoter regulate its activity following interleukin-2 deprivation. (A) Subcellular localization of FoxO3 in CTLL-2 cells. Cells were deprived of IL-2 for 3 hours and then cultured with or without IL-2 for 1 hour. Western blotting was performed using a specific anti-FoxO3 antibody. After stripping, the membrane was reblotted with an anti–β Tubulin antibody to control the possible nuclei contamination with cytoplasmic material. *Shifted band of phosphorylated FoxO3 compared with the unshifted band. Cyto indicates cytoplasmic extract; N, nuclear extract. (B) IL-2 inhibits FoxO3 transcriptional activity. CTLL-2 cells were transiently transfected with 10 μg FHRE-Luc and cultured with or without IL-2 for 8 hours. Results are expressed as a percentage of FHRE-Luc activity and represent the mean ± SEM (error bars) of 2 independent experiments performed in duplicate. (C) Alignment of the consensus core FHRE with the 3 FHREs present in the gilz promoter. The mutations that were introduced for the experiments described in panels D and E are in bold characters. (D) DNA binding of FoxO3 on the various FHREs present in the gilz promoter. The 5′ biotinylated FHRE-1, FHRE-2, and FHRE-3 probes (upper panel) and mutated FHRE-1 and FHRE-3, FHRE-1mut and FHRE-3mut (lower panel) coupled to agarose beads were incubated with nuclear extracts from CTLL-2 cells transiently transfected with pcDNA3-FoxO3. Bound proteins were identified by Western blotting using anti-FoxO3 specific antibody. (E) Mutations of FHRE-1 and FHRE-3 in the gilz promoter affect its regulation. CTLL-2 cells were transiently transfected with the reporter plasmid p-1940 wild type (wt) or with either p-1940FHRE-1mut, p-1940FHRE-3mut, or p-1940FHRE-1+3mut (10 μg) containing one mutation in FHRE-1, one mutation in FHRE-3, or both mutations, respectively. Results are expressed as a percentage of p-1940 activity with 100% being the activity of the wild-type reporter construct in the absence of IL-2. Data represent the mean ± SEM (error bars) of 3 independent experiments performed in duplicate.

Forkhead responsive elements present in the gilz promoter regulate its activity following interleukin-2 deprivation. (A) Subcellular localization of FoxO3 in CTLL-2 cells. Cells were deprived of IL-2 for 3 hours and then cultured with or without IL-2 for 1 hour. Western blotting was performed using a specific anti-FoxO3 antibody. After stripping, the membrane was reblotted with an anti–β Tubulin antibody to control the possible nuclei contamination with cytoplasmic material. *Shifted band of phosphorylated FoxO3 compared with the unshifted band. Cyto indicates cytoplasmic extract; N, nuclear extract. (B) IL-2 inhibits FoxO3 transcriptional activity. CTLL-2 cells were transiently transfected with 10 μg FHRE-Luc and cultured with or without IL-2 for 8 hours. Results are expressed as a percentage of FHRE-Luc activity and represent the mean ± SEM (error bars) of 2 independent experiments performed in duplicate. (C) Alignment of the consensus core FHRE with the 3 FHREs present in the gilz promoter. The mutations that were introduced for the experiments described in panels D and E are in bold characters. (D) DNA binding of FoxO3 on the various FHREs present in the gilz promoter. The 5′ biotinylated FHRE-1, FHRE-2, and FHRE-3 probes (upper panel) and mutated FHRE-1 and FHRE-3, FHRE-1mut and FHRE-3mut (lower panel) coupled to agarose beads were incubated with nuclear extracts from CTLL-2 cells transiently transfected with pcDNA3-FoxO3. Bound proteins were identified by Western blotting using anti-FoxO3 specific antibody. (E) Mutations of FHRE-1 and FHRE-3 in the gilz promoter affect its regulation. CTLL-2 cells were transiently transfected with the reporter plasmid p-1940 wild type (wt) or with either p-1940FHRE-1mut, p-1940FHRE-3mut, or p-1940FHRE-1+3mut (10 μg) containing one mutation in FHRE-1, one mutation in FHRE-3, or both mutations, respectively. Results are expressed as a percentage of p-1940 activity with 100% being the activity of the wild-type reporter construct in the absence of IL-2. Data represent the mean ± SEM (error bars) of 3 independent experiments performed in duplicate.

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