Fig. 5. Differential gene activation by Stat4 isoforms. (A) Spleen cells CD2:Stat4α or CD2:Stat4β were activated with anti-CD3 for 72 h. Cells were then washed and incubated for the indicated time periods with 2 ng/ml IL-12. RNA was isolated, electrophoresed and transferred to nylon membrane. Membranes were hybridized sequentially with radiolabeled IL-12Rβ2, p27Kip1 and SH2-B cDNAs. The blot was stripped and re-probed with TCRα as a control for loading. Densitometry was performed following autoradiography, and expression was normalized to TCRα expression and is presented as the fold induction over expression of the genes in unstimulated cells. Results are representative of several different northern analyses. (B) Autoradiographs of northern analysis of the induction of genes analyzed in (A). (C) Western analysis of p27Kip1 levels in Stat4α and Stat4β transgenic Th1 cells. Protein extracts from cells incubated with or without 2 ng/ml IL-12 for the indicated times were resolved by SDS–PAGE and immunoblotted with monoclonal anti-p27Kip1 followed by stripping and probing with anti-GAPDH as a loading control. Results are representative of two experiments.