Expression and phosphorylation of Stat6. (A) Naive TG and WT CD4⁺ CD62L^high T cells were cultured with anti-CD3/anti-CD28 Abs in the presence or in absence of anti–IL-4 Ab (10 μg/mL) for the indicated times. Protein extracts were processed for immunoblot analysis using anti-Stat6 Ab (top lane) or anti–phospho-Stat6 (pStat6) (middle lane). Quantitative analysis of Stat6/β-tubulin and pStat6/β-tubulin ratio is shown. Data shown are representative of 2 independent experiments. Quantitative analysis of Stat6/GAPDH, as evaluated by RT-PCR, is shown; bars represent the mean values ± 1 SEM of 2 independent experiments. ^**P < .01, TG vs WT. (B) CD4⁺ cells were transfected with siRNAs specific for Stat6 and, 1 hour later, activated by anti-CD3/anti-CD28 Abs for 24 hours. Western blot was analyzed by specific Abs for Stat6 and, after stripping for GATA-3 and β-tubulin. Quantitative analysis of Stat6/β-tubulin and GATA-3/β-tubulin is shown.