Expression of GATA-3 and T-bet. (A) Naive TG and WT CD4⁺CD62L^high T cells were cultured with anti-CD3/anti-CD28 Abs for the times indicated. On day 7, cells were washed and restimulated for 6 hours with anti-CD3 Ab. Protein extracts from freshly naive T cells (day 0) and cultured cells (days 1 and 7) were processed for immunoblot analysis using anti–GATA-3 Ab. The same extracts were re-probed with anti–T-bet and anti–β-tubulin Abs. Quantitative analysis of GATA-3/β-tubulin, T-bet/β-tubulin, and GATA-3/T-bet ratios are shown. (B) GATA-3 and T-bet expression, evaluated by real-time quantitative PCR, in CD4⁺CD62L^high freshly isolated or cultured with anti-CD3/anti-CD28 Abs for the indicated times. Relative expression is shown. Data are the mean values ± 1 SEM. ^*P < .05, TG vs WT. (C) GATA-3 and T-bet expression (as evaluated by Western blot analysis) in freshly isolated CD4⁺ T cells from regional lymph nodes of OVA-immunized mice or control mice. Western blot with β-tubulin was performed to ensure that equivalent amounts of proteins were loaded in each lane. Data are representative of 2 independent experiments. (D) The BW5147 cell line was transfected with pcDNA3-GILZ or pcDNA3 empty vector. Western blot analyses of GILZ and β-tubulin proteins are shown with the histogram of the GILZ/β-tubulin ratio (Di). mRNA expression was measured by semi-quantitative RT-PCR for GILZ (Dii), GATA-3 (Diii), and IL-13 (Div). RT-PCR data were normalized by 1D image analysis software to GAPDH expression and presented as mean ± 1 SEM of duplicate samples from 3 independent experiments. ^*P < .05 GILZ-transfected vs empty-vector–transfected cells.