Fig. 7. Defective Stat1 activation and serine phosphorylation in PKR-null cells. (A) 8× con GAS linked to luciferase was transiently transfected into wild-type or PKR-null MEFs. Luciferase activity was determined with or without murine IFN–γ (1000 IU/ml) for 6 h. Results are presented with standard deviations from three independent experiments. (B) Whole-cell extracts were prepared from serum-starved wild-type or PKR-null cells, with or without treatment with 1000 IU/ml of murine IFN–γ. Stat1 binding to the SIE (m67) GAS was determined by EMSA. (C) Extracts of serum-starved cells, either untreated or treated with IFN–γ (1000 IU/ml for 20 min) were immunoprecipitated with anti-Stat1. The transfer was probed first with an antibody specific for a Stat1 peptide that includes phosphorylated Ser727 and then reprobed with anti-Stat1. (D) RNA from MEFs untreated or treated with murine IFN–γ (1000 IU/ml) was analyzed by the Northern procedure.