Nuclear translocation and transcriptional activity of stably transfected cells. Stably transfected U-266-1970-pcIneo cells and U-266-1970-Stat1C cells were left un-treated or were treated with 1000 U/mL IFN-γ for 0.5 hours. Nuclear and cytoplasmic protein extracts were prepared. ( A) Western blot analysis of the nuclear (n) and cytoplasmic (c) protein extracts were performed using the indicated antibodies. The proteins α-tubulin and α-histone H3 are markers for the cytoplasmic and nuclear fraction respectively. ( B) Stably transfected U-266-1970-pcIneo or U-266-1970-Stat1C cells were left untreated or were treated with IFN-γ (1000 U/mL) for the indicated times. Protein lysates were prepared and Western blot analysis was performed using the indicated antibodies. ( C) The luciferase reporter contruct GBP-luc was cotransfected with hubactp/lacZ vector into U-266-1970-pcIneo cells and U-266-1970-Stat1C cells as described. After 24 hours of treatment with IFN-γ (1000 U/mL), protein extracts were prepared and luciferase assays were performed. The graph shows fold induction of relative light units (RLU) as the mean of three independent experiments ± SD. Corrections have been made for varying transfection efficiency.