Fig. 3.

PML inhibits DNA binding activity of STAT-1α. (A) Nuclear extracts from immortalized Pml^+/+ and Pml^−/− MEF treated with IFN-γ for 0–5 h were incubated in the presence of a radiolabeled probe containing a GAS element and subjected to EMSA. Anti-STAT-1α or -STAT-3 antibodies were added to test the specificity of interaction. Binding specificity was also tested by adding a 100-fold molar excess of cold GAS probe (competition). (B) Cells were treated with IFN-γ for up to 4 h and fixed by using formaldehyde. Chromatin was sheared and immunoprecipitated by using STAT-1 antibody or normal rabbit IgG, then amplified by RT-PCR using primers designed for the murine CIITA pIV promoter. (C) Nuclear extracts from Pml^−/− MEF treated with IFN-γ for 1 h were preincubated with PML IV protein (0–100 ng) for 1 h and then subjected to EMSA (lanes 1–3). BSA was used as a control for the PML protein (lane 4). Representative of three experiments.