Figure 3
Figure 3. FoxP3 interacts with c-Jun. (A) Xpress-tagged c-Jun Myc-FoxP3 expression plasmids were cotransfected into HEK 293 cells. Transfected cells were collected and lysed 48 hours after transfection. FoxP3 in the cell lysates was immunoprecipitated with anti-Myc antibody. The bound c-Jun was detected by Western blotting using anti-Xpress antibody (top panel). The same membrane was reprobed with anti-FoxP3 antibody (second panel from top). As controls, the levels of c-Jun and Actin in the whole cell lysates were determined with Western blotting using anti-Xpress and Actin, respectively (bottom 2 panels). (B) CD4+ CD25+ T cells were isolated and stimulated with anti-CD3 plus anti-CD28 or PMA plus ionomycin for 30 minutes. Stimulated cells were lysed, and FoxP3 in the cell lysates was immunoprecipitated with anti-FoxP3 antibody; the bound c-Jun in the immunoprecipitates was detected with anti-c-Jun antibody (top panel). The same membrane was reprobed with anti-FoxP3 antibody (second panel from top). As controls, the protein levels of c-Jun and Actin in the whole cell lysates were determined with Western blotting using anti-c-Jun and anti-Actin antibodies, respectively (bottom 2 panels). (C) Xpress-tagged c-Jun and Myc-tagged FoxP3 mutants, FN/NLS or FHK, expression plasmids, were cotransfected into HEK 293 cells. FoxP3 mutant proteins in these lysates from transfected cells were immunoprecipitated with anti-Myc antibody, and the bound c-Jun was detected with anti-Xpress antibody (top panel). The same membrane was reprobed with anti-Myc antibody for detecting the expression of FoxP3 mutants (second panel from top). As controls, the protein levels of c-Jun and Actin were determined by Western blotting with anti–c-Jun and anti-Actin, respectively (bottom 2 panels). (D) AP-1-luciferase and c-Jun expression plasmids were cotransfected without or with FoxP3 or with each of FoxP3 mutants. The luciferase activities in the cell lysates were detected. Error bars represent data from 3 independent experiments. (E) HEK293 cells were transfected with empty plasmid or with plasmid encoding full-length FoxP3 (FoxP3 FL) or each of FoxP3 truncated mutants (FN/NLS and FHK) as indicated. Nuclear extracts were isolated 48 hours after transfection. Equal amounts of nuclear proteins were incubated with biotin-labeled AP-1 promoter fragments and gel shift assay was performed.

FoxP3 interacts with c-Jun. (A) Xpress-tagged c-Jun Myc-FoxP3 expression plasmids were cotransfected into HEK 293 cells. Transfected cells were collected and lysed 48 hours after transfection. FoxP3 in the cell lysates was immunoprecipitated with anti-Myc antibody. The bound c-Jun was detected by Western blotting using anti-Xpress antibody (top panel). The same membrane was reprobed with anti-FoxP3 antibody (second panel from top). As controls, the levels of c-Jun and Actin in the whole cell lysates were determined with Western blotting using anti-Xpress and Actin, respectively (bottom 2 panels). (B) CD4+ CD25+ T cells were isolated and stimulated with anti-CD3 plus anti-CD28 or PMA plus ionomycin for 30 minutes. Stimulated cells were lysed, and FoxP3 in the cell lysates was immunoprecipitated with anti-FoxP3 antibody; the bound c-Jun in the immunoprecipitates was detected with anti-c-Jun antibody (top panel). The same membrane was reprobed with anti-FoxP3 antibody (second panel from top). As controls, the protein levels of c-Jun and Actin in the whole cell lysates were determined with Western blotting using anti-c-Jun and anti-Actin antibodies, respectively (bottom 2 panels). (C) Xpress-tagged c-Jun and Myc-tagged FoxP3 mutants, FN/NLS or FHK, expression plasmids, were cotransfected into HEK 293 cells. FoxP3 mutant proteins in these lysates from transfected cells were immunoprecipitated with anti-Myc antibody, and the bound c-Jun was detected with anti-Xpress antibody (top panel). The same membrane was reprobed with anti-Myc antibody for detecting the expression of FoxP3 mutants (second panel from top). As controls, the protein levels of c-Jun and Actin were determined by Western blotting with anti–c-Jun and anti-Actin, respectively (bottom 2 panels). (D) AP-1-luciferase and c-Jun expression plasmids were cotransfected without or with FoxP3 or with each of FoxP3 mutants. The luciferase activities in the cell lysates were detected. Error bars represent data from 3 independent experiments. (E) HEK293 cells were transfected with empty plasmid or with plasmid encoding full-length FoxP3 (FoxP3 FL) or each of FoxP3 truncated mutants (FN/NLS and FHK) as indicated. Nuclear extracts were isolated 48 hours after transfection. Equal amounts of nuclear proteins were incubated with biotin-labeled AP-1 promoter fragments and gel shift assay was performed.

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