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Blood, Vol. 111, Issue 7, 3599-3606, April 1, 2008
FoxP3 maintains Treg unresponsiveness by selectively inhibiting the promoter DNA-binding activity of AP-1
Blood Lee et al.
111: 3599
Supplemental materials for: Lee et al
Files in this Data Supplement:
- Table S1. Probes used in EMSA assays (PDF, 35.1 KB)
- Figure S1. Interaction of FoxP3 with AP-1 family proteins (JPG, 47.9 KB)
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Myc-tagged FoxP3 expression plasmids were transfected into HEK 293 cells. (A) Cell lysates were used for immunoprecipitation with normal IgG of rabbit or with antibodies against c-Fos, JunB and ATF-2, respectively. The immunoprecipitates were subjected onto SDS-PAGE and western blotting with anti-Myc antibody. (B) The expression levels of FoxP3 (top panel), c-Fos (2nd panel), JunB (3rd panel), ATF-2 (4th panel) and Actin (bottom panel) in the whole cell lysates were analyzed by western blotting.
- Figure S2. Subcellular localization of FoxP3 mutants (JPG, 54.8 KB)
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HEK293T cells were transfected with Myc-tagged FoxP3 mutants including 1-coil, 1-coil/NLS or FH expression plasmid. 36 hrs after transfection, cells were immunostained with anti-Myc antibody followed by second antibody labeled with Alexa-594. Nuclear DNA was stained with DAPI (blue). Cells were then visualized under the fluorescence microscopy.
- Figure S3. FoxP3 expression alters c-Jun subnuclear localization (JPG, 56.5 KB)
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(A) c-Jun-CFP expression plasmids were transfected without or with FoxP3 48 hours after transfection, 200 cells with cyan fluorescence of each transfection were counted. The percentages of doted verses diffused nuclear distributions were calculated. (B) c-Jun-CFP expression plasmids were transfected without or with various amounts of FoxP3 expression plasmids into HEK 293 cells. 48 hours after transfection, 200 cells with cyan fluorescence of each transfection were counted. The percentages of doted verses diffused nuclear distributions were calculated.
- Figure S4. Imaging FoxP3/c-Jun interaction by BiFC (JPG, 41.4 KB)
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YN-FoxP3 and c-Jun-CC expression plasmids were co-transfected into HEK 293 cells. Transfected cells were cultured for 36 hours at 37°C followed by cultivating at 30°C for another 12-16 hours. Cells were fixed and stained with DAPI for nuclear DNA, and then visualized by fluorescence microscope.
- Figure S5. ChiP analysis of c-Jun binding at IL-2 promoter (JPG, 43.9 KB)
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(A) ChIP assays were performed on chromatin isolated from CD25- T cells and Tregs cells before and after CD3/28 stimulation (24hrs). Antibodies for c-Jun were used for immunoprecipitation and ChiP extracts were probed for the regulatory region of IL-2 promoter by PCR. (B) Total DNA from these cells in (A) was analyzed by PCR as input controls.
- Figure S6. Inhibition of FoxP3 expression by siRNA (JPG, 40.8 KB)
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(A) The 19-mer nucleotides were selected for FoxP3 targeting. (B) CD4+CD25+ Tregs were purified and infected with lentiviruses that carry empty pLL3.7 vector or pLL3.7-FoxP3. GFP+ cells were sorted and lysed. The cell lysates and its 1:3 dilutions were subjected to western blotting analysis of FoxP3 expression (top panel). The same membrane was reprobed by anti-Actin as a control (bottom panel).
- Figure S7. Effects of BLP on the protein expression of FoxP3 in Tregs (JPG, 56 KB)
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(A) CD4+CD25+ T cells were isolated and stimulated with anti-CD3 plus anti-CD28 in the absence or presence of BLP for one hour. The expression levels of FoxP3 protein was detected by western blotting using anti-FoxP3 antibody (top panel). The same membrane was reprobed with anti-c-Jun (middle panel) and anti-Actin (bottom panel), respectively. (B) FoxP3 expression plasmids were transfected into 293 cells. Cells were treated with cycloheximide (50 mg/ml) for different amount of time (chase time). The protein levels of FoxP3 were analyzed by anti-FoxP3 antibody (top panel). The same membrane was reblotted with anti-Actin as a control (bottom panel). (C) The band densities of FoxP3 western in (B) were quantified. Error bars represent data from three independent experiments.
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