Impaired c-Maf expression in Vav1^–/– Th2 cells is linked to deficient Ca²⁺ signaling. (A) T cells were stimulated under Th2-inducing conditions in the presence or absence of the indicated concentrations of ionomycin. After 7 days, the cells were washed, counted, and restimulated in anti-CD3–coated tissue culture plates. Proliferation (left) and cytokine-producing cells (right) were determined as in Figure 2. Data are expressed as the mean value of triplicate determinations ± SE. (B) Naive CD4⁺ T cells were cultured under Th2- or Th1-inducing conditions in the presence or absence of the indicated ionomycin concentrations. Lysates prepared 48 hours after restimulation were analyzed by immunoblotting. Numbers indicate the intensity of c-Maf signal relative to Vav1^–/– Th2 cells cultured without ionomycin (= 1) after normalization to the Grb2 signal. (C) Naive CD4⁺ T cells from wild-type mice were cultured under Th2-inducing conditions in the presence or absence of indicated doses of cyclosporin A. Lysates prepared 48 hours after restimulation were analyzed by immunoblotting. The c-Maf signal was quantitated as in panel B. (D) Primary CD4⁺ T cells were stimulated with IL-4 (100 U/mL) for 5 minutes and cell lysates resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were immunoblotted with the indicated antibodies.