FIGURE 7. FoxP3 alters NFκB occupancy to the LTR promoter.
A, FoxP3 expression does not affect NFκB localization in the nucleus. Vector control and FoxP3 transduced Jurkat T cells were fractionated, and cytoplasmic and nuclear lysates were separated by SDS-PAGE. Membrane was probed for NFκB using anti-p65 antibody. B, FoxP3 does not alter NFκB p65 and p50 binding to the MHC II-derived consensus NFκB probe. Nuclear extract of transduced Jurkat T cells were left unstimulated or PMA/ionomycin stimulated for 30 min, and NFκB binding was assessed. Antibodies specific for p65 and p50 were used for supershift. C and D, Jurkat T cells with a stably integrated HIV-1 LTR were transduced with control or FoxP3-expressing vectors. Sonicated chromatins were immunoprecipitated with control IgG or anti-p65 antibody. Samples were purified and subjected to real-time qPCR analysis. Input chromatin (10% relative to each immunoprecipitation) was used to normalize samples. The relative p65 occupancy at the HIV-1 LTR (C) or IL-2 promoter (D) from a representative experiment is shown as relative anti-p65-specific ChIP signals divided by signals from control input chromatins (**, p < 0.01).