Figure 7. Ndfip1 expression peaks early during iT_reg cell differentiation to attenuate JunB expression.

(a) qRT-PCR analysis of Ndfip1 expression. Results presented are relative to the expression of Ndfip1 in naïve T cells (RQ) and show one representative plot (n=7, 3 independent experiments) (mean + s.d. of triplicate samples). (b and c) JunB protein (normalized to GAPDH) at day 3 of iT_reg cell conversion. Values show fold expression over WT (set to one and indicated by the dashed gray line) (b) Representative blot and (c) graph of cumulative data from Ndfip1^+/+ (Wt), Ndfip1^–/– (N) and Itchy mutant (I) iT_reg cells (mean + s.d., n≥3). (d and e) c-Jun (d) and JunD (e) are shown as described above for JunB. (f) ChIP analysis of T cells containing (+) or lacking (-) Ndfip1. PCR using primers specific for the Il4 promoter was performed on chromatin DNA obtained before (Input) and after immunoprecipitation (IP) with anti-JunB or control IP. (n=5 Ndfip1^+/+ and n=4 Ndfip1^–/–. 2 independent experiments) (g-h) Representative blot of JunB expression in T cells containing (+/+) or lacking (–/–) Ndfip1 after approximately 2 days of stimulation (g) under normal iT_reg conditions (+) or in the absence (-) of TGF-β and (h) under normal iT_reg cell conditions (TCR+) or under TCR withdrawal conditions (TCR-) described in supplementary materials. Representative blot of JunB expression is shown. Values shown are adjusted for GAPDH and normalized to Ndfip1^+/+ T cells (set to 1) stimulated +TGF-β (g) or normal iT_reg cell conditions (+TCR) (h).