Figure 2. Activation in the presence of IL-4 and IL-12 results in the maintenance of the IL-12 signaling pathway. FACS®-sorted naive CD4+ DO11.10 T cells were cultured with OVA peptide and irradiated BALB/c splenocytes in the presence of 10 U/ml IL-12 and 10 μg/ml anti–IL-4 to promote Th1 differentiation (upper), 10 U/ml IL-12 and 200 U/ml IL-4 (middle), or 200 U/ml IL-4 and 3 μg/ml anti–IL-12 to promote Th2 differentiation (bottom). After 72 h the cells were expanded 10-fold in fresh medium containing 40 U/ml IL-2. On day 5 after primary antigen activation whole cell lysates from developing T cells (1.5–2.0 × 107) were prepared after incubation for 25 min with medium alone or with 10 U/ml IL-12. Lysates were immunoprecipitated with Stat4 antiserum, separated by SDS-PAGE (7% gel), transferred to nitrocellulose, and probed with the anti-phosphotyrosine reagent RC20 as described (30). After exposure, blots were stripped and reprobed with anti-Stat4 antisera.
Figure 2

Activation in the presence of IL-4 and IL-12 results in the maintenance of the IL-12 signaling pathway. FACS®-sorted naive CD4+ DO11.10 T cells were cultured with OVA peptide and irradiated BALB/c splenocytes in the presence of 10 U/ml IL-12 and 10 μg/ml anti–IL-4 to promote Th1 differentiation (upper), 10 U/ml IL-12 and 200 U/ml IL-4 (middle), or 200 U/ml IL-4 and 3 μg/ml anti–IL-12 to promote Th2 differentiation (bottom). After 72 h the cells were expanded 10-fold in fresh medium containing 40 U/ml IL-2. On day 5 after primary antigen activation whole cell lysates from developing T cells (1.5–2.0 × 107) were prepared after incubation for 25 min with medium alone or with 10 U/ml IL-12. Lysates were immunoprecipitated with Stat4 antiserum, separated by SDS-PAGE (7% gel), transferred to nitrocellulose, and probed with the anti-phosphotyrosine reagent RC20 as described (30). After exposure, blots were stripped and reprobed with anti-Stat4 antisera.

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