Figure 2  FKH Domain of Foxp3 Is Required to Inhibit NF-κB Activation in HEK 293T Cells but Not CD4+ T Cells
(A) Schematic representation of human Foxp3, including proline-rich (P-P-P), zinc finger (ZNF), leucine zipper (ZIP), and forkhead (FKH) domains. Mutations within the Foxp3 gene associated with IPEX have been previously described [4,14,43]. The FKH domain contains a nuclear localization signal (NLS), which is required for Foxp3 expression in the nucleus.
(B and C) HEK 293T cells (5 × 105) (B) were seeded into six-well plates 1 d prior to transfection or Jurkat T cells (1 × 106) (C) were seeded into six-well plates on the day of transfection with an NF-κB luciferase reporter vector (200 ng) in the presence or absence of a Foxp3, ΔFKH, or control expression vector (1,000 ng) and internal reference plasmid (pGL4-TKhRluc2; 50 ng). Cells were harvested at 24 h posttransfection and analyzed for luciferase activity. Relative luciferase activity was normalized to the internal reference control.
(D) CD4+ T cells (2 × 106) from healthy donors were nucleofected with an NF-κB reporter plasmid (2,000 ng), together with either a Foxp3, ΔFKH, or control expression vector (2,000 ng), and an internal reference plasmid (1,000 ng). Cells were harvested 24 h posttransfection and analyzed for luciferase activity. Relative luciferase activity was normalized to the internal reference control. Results from one healthy donor are shown.
(E) Foxp3, ΔFKH, and NF-κB p65 expression in whole-cell extracts (20 μg) derived from HEK 293T cells transfected with a control expression vector (EGFP), Foxp3, or ΔFKH expression vectors were analyzed by Western blot analysis. β-actin expression was analyzed as a loading control.