Fig. 1. Eos is highly expressed in subpopulation of CD4+ T cells, and it physically interacts with Foxp3 in Treg cells.
CD4+ T cells pooled from spleens and LN of C57BL/6 or GFP-Foxp3 mice were sorted into indicated subsets, and the expression of Eos was assessed by (A) real-time quantitative RT-PCR analysis using primers and internal fluorescent probes specific for Eos and 18s rRNA, and (B) Western blot analysis, respectively. (C) Endogenous interaction between Eos and Foxp3 in Treg cells. Treg cells were lysed, subjected to immunoprecipitation (IP) with anti-Foxp3 (left), anti-Eos (right) antibodies or control IgG. The immunoprecipitates were resolved on SDS-PAGE followed by Western blot analysis (WB) with anti-Eos and anti-Foxp3 antibodies as indicated, respectively. (D) In vitro interaction between Eos and Foxp3 was analyzed by GST pull-down assay. In vitro transcribed/translated [35S]-methionine-labelled Eos or Foxp3 was incubated with glutathione beads bound with bacterially expressed GST-Foxp3 or GST-Eos, respectively. GST alone was included as a negative control. After 30 mins incubation, the beads were washed 3 times with PBS and eluted with 5mM reduced glutamine. The eluted product was subjected to SDS-PAGE and autoradiograph. Input consisted of 10% of the [35S]-methionine-labelled proteins. (E) Coomassie blue staining. GST fusion protein purified from bacterially expressed GST-Foxp3 or GST-Eos were subjected to SDS-PAGE and stained with Coomassie blue. (F) Mapping of the Eos-interacting domain in Foxp3 with a mammalian two-hybrid system. The cDNA of Eos was fused to the VP16 AD in the pVP16 vector (Clontech), the cDNAs of different fragments of Foxp3 were fused to GAL4 DB in the pM vector. 293T cells were transfected with the indicated vectors together with pnull-RL (Renilla Luciferase, Promega) and pG5Luc, a luciferase reporter plasmid containing GAL4 promoter. After 24h, cells were harvested and luciferase activity was measured and normalized to Renilla luciferase activity. (G) Mapping of the Eos-interacting Domain in Foxp3 by coimmunoprecipitation. 293T cells were transfected with the indicated plasmids. After 24hr, cell extracts were immunoprecipitated with anti-DBD antibodies (Santa Cruz), the immune complexes were subjected to SDS-PAGE, and blotted with anti-Flag (top), anti-DBD (middle). The whole cell lysates were blotted with anti-Flag antibody (bottom). (H) The co-IP assay was applied to map the Eos-interaction domain in Foxp3. Numbers at top of each lane represent the peptide fragments of Foxp3 fused to the DBD epitope. (I) The co-IP assay was applied to map the Foxp3-interaction domain in Eos. Numbers at top of each lane represent the peptide fragments of Eos fused to the Myc epitope. The results are representative of one of three independent experiments.