IFN-γ restores the IL-12 signaling pathway in developing Th2 cells. FACS^®-sorted naive CD4⁺ DO11.10 T cells were cultured with OVA peptide and irradiated BALB/c splenocytes under the conditions indicated. In the primary cultures, IFN-γ levels were either unaltered (none), neutralized with 30 μg/ml of the anti–IFN-γ mAb H22 (anti–IFN-γ) or 100 U/ml of IFN-γ (IFN-γ) were added as indicated. On day 5 after primary antigen activation whole cell lysates from developing T cells (20 × 10⁶) were prepared after incubation for 25 min 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. After exposure, blots were stripped and reprobed with anti-Stat4 antisera.