Viperin is required for optimal GATA3 induction. (A) Total-cell lysates were prepared from activated CD4⁺ T cells and blotted with anti–GATA-3 and anti–T-bet. Tubulin-α was used as a loading control. (B) Immunofluorescence staining of GATA-3 (green) and CD4 (red) in stimulated T cells, counterstained with Topro-3 (purple). (C) T-bet and Gata3 mRNA expression by CD4⁺ T cells in response to stimulation at indicated times. (D) RT-PCR analyses of naive CD4⁺ T cells cultured under suboptimal Th2-skewed conditions consisted of immobilized 5 μg/mL anti-CD3 and 1 μg/mL anti-CD28, exogenous IL-2, and the indicated amount of IL-4 for 5 days. Data (C,D) are expressed as the mean value of triplicate determinations plus or minus SD, and the data shown are representative of 3 independent experiments. *P < .05; **P < .01, WT versus KO. (E,F) Cytosolic and nuclear extracts prepared from CD4⁺ T cells stimulated as indicated for 72 hours (E) or for 4 days (F) were processed for immunoblotting. β-actin was used as a control for equal protein loading. A total of 4 (E) or 3 (F) independent experiments were performed with similar results. (G) Indo-1–loaded CD4⁺ T cells were subjected to challenge with anti-CD3 (5 μg/mL), goat–anti-hamster IgG (40 μg/mL; GAH), and ionomycin (500 ng/mL; iono) sequentially. Histogram data are presented as median ratio of calcium mobilization as measured by fluorescence-activated cell sorter (FACS). Experiments were repeated twice with similar results. (H) Nuclear translocation of NFATc-1 and NFATc-2 was analyzed with extracts prepared from T cells stimulated for 72 hours. Data are representative of 3 independent experiments.