(A-B) 293ET cells were transfected with 400 ng of (A) m5p[FLAG-p65] or (B) m5p[FLAG-c-Rel] together with 50 ng of the NF-κB-reporter (firefly) m3p-luc[NF-κB], 20 ng pRL-TK and increasing amounts of P8[HA-Foxp3] or GFP as a control gene (P8[control]) as indicated. Forty-eight hours later, the cells were analyzed for NF-κB activity, which was normalized against the renilla luciferase signal. Transfections were performed in duplicate wells and two samples were measured in each case (*** = p<0.001, ANOVA). (C) 293ET cells were co-transfected with m5p[luc-c-Rel] and m5p[FLAG-Foxp3] or GFP as a control gene m5p[FLAG-control]. Forty-eight hours later cytoplasmic and nuclear cell lysates were prepared and the interaction between the two proteins were measured using LUMIER assays. (D) 293ET cells were co-transfected with m5p[luc-c-Rel] and m5p[FLAG-Foxp3^mut] or FLAG-GFP as a control gene m5p[FLAG-control]. Forty-eight hours later total cell lysates were prepared and used for LUMIER assays. (A–D) In each case the experiment was repeated three times and a representative example is shown. The error-bars represent the SEM.

(A–C) 293ET cells were co-transfected with m5p[FLAG-Foxp3^ΔFKH] and various m5p[luc-c-Rel^mut] as shown in the schematic on the left. Forty-eight hours later total cell lysates were prepared and used in LUMIER assays. Cell lysates were precipitated using anti-FLAG agarose. (A) Fold binding indicates the ratio of luciferase activity in precipitates and cell lysates normalized to a control (GFP) reaction. (B) Total cell lysates were analyzed by Western Blots (insert) for the expression of the FLAG-tagged Foxp3^ΔFKH protein. (C) Total luciferase activity in lysate to show the expression of luc-c-Rel^mut. A representative example of three independent experiments is shown. The error-bars represent the SEM within the experiment.

(A) 293ET cells were transfected with 400 ng m5p[c-Rel], 50 ng m3p-luc[NF-κB], 20 ng pRL-TK and increasing amounts of P8[Foxp3] or P8[Foxp3^Δe1]. Two independent experiments were performed and fold NF-κB activation was pooled and normalized against the highest signal. The error-bars represent the SEM, (*** = p<0.001; two-way ANOVA). (B) Alternatively, the cells were transfected with 50 ng m3p-luc[NF-κB], 20 ng pRL-TK and 400 ng P8[control] (GFP), P8[Foxp3] or P8[Foxp3^ΔFKH] in the presence or absence of 400 ng m5p[c-Rel]. Forty-eight hours later, the cells were analyzed for NF-κB activity, which was normalized against the renilla luciferase signal (*** = p<0.001; unpaired t test). (C-E) 293ET cells were co-transfected with m5p[GFP-Foxp3^ΔFKH] in the (C) absence or (D) presence of m5p[c-Rel] and analyzed by confocal microscopy after 48 h. (E) Pooled data from two independent experiments where 293ET cells were co-transfected with m5p[GFP-Foxp3^ΔFKH] and rel-homology domain deletion (c-Rel^168-588) or transactivation domain deletion (c-Rel^1-287) of c-Rel. For each of the conditions, several randomly picked field of visions (∼10 cells) were scored for the presence of Foxp3^ΔFKH in the nucleus. As a control, cells were transfected with rCD8α (m5p[control]) (*** = p<0.001; unpaired t test).

293ET cells were co-transfected with m5p[luc-c-Rel] and various m5p[FLAG-Foxp3^ΔFKH:mut] as shown in the schematic on the left. Forty-eight hours later total cell lysates were prepared and used in LUMIER assays. Cell lysates were precipitated using anti-FLAG agarose. Fold binding indicates the ratio of luciferase activity in precipitates and cell lysates normalized to a control (GFP) reaction. Total cell lysates were analyzed by Western Blots (insert) for the expression of the FLAG-tagged Foxp3^ΔFKH:mut proteins. Foxp3^{ΔFKH: Δe5} is only expressed at very low levels (arrow). A representative example of three independent experiments is shown. The error-bars represent the SEM within the experiment. ZnF  =  zinc finger, CC  =  coiled-coil.

(A–C) 293ET cells were co-transfected with m5p[FLAG-NFAT] and various m5p[luc-Foxp3^mut] as shown in the schematic on the left. Forty-eight hours later total cell lysates were prepared and used in LUMIER assays. Cell lysates were precipitated using anti-FLAG agarose. (A) Fold binding indicates the ratio of luciferase activity in precipitates and cell lysates normalized to a control (GFP) reaction. (B) Total cell lysates were analyzed by Western Blots for the expression of the FLAG-tagged NFAT proteins. (C) Total luciferase activity in lysate to show the expression of luc-Foxp3^mut. A representative example of three independent experiments is shown. The error-bars represent the SEM within the experiment.