IFN-γ–dependent Stat1, Stat1K703R, and Stat1E705A activation. (A) Stat1^–/– MEFs, infected with an empty vector (pMIG) or retroviral vectors directing expression of either Stat1 (St1), Stat1^k703R (St1^K703R), or Stat1^E705A (St1^E705A), were subsequently transfected with HA–SUMO-1 and Ubc9 cDNA constructs. WCEs were prepared, immunoprecipitated with anti-Stat1, fractionated on 7% SDS-PAGE, and immunoblotted for Stat1 (top panel); or extracts were directly fractionated on 12% SDS-PAGE and sequentially immunoblotted with anti-Stat1 and anti-HA (bottom panels). WCEs from 293T cells transfected with HA–SUMO-1 and Ubc9 from Figure 1 served as positive controls. (B) Stat1^–/– MEFs from panel A were stimulated with IFN-γ (50 U/mL for 0.5-12 hours). WCEs were prepared, fractionated by SDS-PAGE, and sequentially immunoblotted with antibodies specific for phosphotyrosine-Stat1 (top panel) and total Stat1 (bottom panel). (C) Extracts from panel B were evaluated by EMSA with a GAS probe. Data are representative of 3 independent studies in MEFs.