Fig 2.

IL-12 signaling is restored in STAT4-deficient splenocytes transduced with wild-type and mutant versions of STAT4. (A) Retroviral constructs encoding wild-type (wt) and Y693F and S721A STAT4 mutants. LTR, long terminal repeat. IRES, internal ribosomal entry site. (B) Tyrosine and serine phosphorylation of wild-type and mutant versions of STAT4 in reconstituted STAT4^−/− spleen cells. Reconstituted spleen cells were serum-starved and stimulated with IL-12 for 45 min. The cells were lysed and immunoprecipitated with anti-STAT4. The immunoprecipitates were electrophoresed and immunoblotted with an antibody that recognizes phosphorylated Y693 (pYSTAT4), pS, and anti-STAT4. IP, immunoprecipitation; IB, immunoblotting. (C) IL-12-dependent proliferation of STAT4^−/− spleen cells reconstituted with wild-type (wt) or mutant STAT4. Transduced spleen cells were stimulated with indicated doses of IL-12 for 24 h, and proliferative responses were determined.