Miz-1 is required for proper IL-7/IL-7R signaling and binds to the SOCS1 promoter. (A) Intracellular pSTAT5 detection in WT and Miz-1ΔPOZ thymocytes after ex vivo stimulation with IL-7. Histograms show isotype control (ctrl) antibodies staining in gray, and pSTAT5 antibody stainings in unstimulated (No IL-7) and stimulated with IL-7 (+ IL-7) cells. Mean fluorescence intensities ± SD are indicated; n = 4 for DN1 and DN2 and n = 3 for CD4−CD8−TCR-γδ+. (B) Total STAT5 proteins in DN1, DN2, and DN3 cells. Cells were sorted and whole protein extracts were evaluated by Western blot for STAT5 (top blot) and β-actin loading control (bottom blot; n = 2). (C) Bcl-2 detection in WT and Miz-1ΔPOZ thymocytes after ex vivo stimulation with IL-7. Mean fluorescence intensities ± SD are indicated. (D) ChIP analysis to identify Miz-1 binding to potential sites within SOCS1 promoter. Cells were rested at 37°C in phosphate-buffered saline for 1 hour, and ChIP was performed on primary DN cells (E) or on SCID.adh murine thymic lymphoma cells (F). Quantitative real-time PCR was performed using primers flanking the initiator region (SOCS1_3) or upstream (SOCS1_5 and _7) of SOCS1 promoter (indicated as arrows in panel D and described in supplemental Table 3). Data are fold enrichment of specific anti–Miz-1 ChIP over rabbit control Ig ChIP (set as 1-fold) from triplicates ± SD (n = 4). *P ≤ .05. **P ≤ .01.
