Figure 1.

IFN-γ production induced by anti-CD3 stimulation in differentiated Th1 cells is IL-12- and Stat4-indepenent. (A) 3F6 T cells were stimulated (+) with anti-CD3 or IL-12 (10 units/ml) as indicated or left unstimulated (−) for 4 hr as described in Methods. Total RNA was prepared and Northern blot analysis for IFN-γ was performed (17). The autoradiogram was overexposed to allow detection of the faint band present in the IL-12-treated lane. The blot was stripped and reprobed for GAPDH as a loading control as described (17). (B) 3F6 T cells were pretreated with Genistein (50 μg/ml) (gen) or cycloheximide (10 μg/ml) (CHX) or left untreated (φ), as indicated, for 30 min and then stimulated with IL-12 (10 units/ml) or anti-CD3 for 30 min, or untreated (none) as indicated. Cells were lysed, and immunoprecipitation for Stat4 was performed by using NB34 as described (17). Western detection of phosphotyrosine was performed with antiphosphotyrosine RC20 (Transduction Laboratories), and blots were stripped and reprobed with anti-Stat4 (Lower) to indicate uniform Stat4 immunoprecipitation.