pY701-STAT1 is the major form of ubiquitinated-STAT1 induced by type-I IFN, and ubiquitin-proteasome degradation controls pY701-STAT1 levels largely.

(A) 293T cells were treated with IFNα (1,000 IU/ml) as indicated following with MG132 (10 μM) pretreatment for 1 hr. STAT1 proteins were immunoprecipitated and analyzed by indicated antibodies. (B) 293T cells were stimulated by IFNα (1,000 IU/ml) for 15 min. pY701-STAT1 proteins were separated by immunoprecipitation using pY701 antibody. The supernatant from pY701-STAT1-immunoprecipitation was subjected to STAT1 immunoprecipitation using STAT1 antibody. Then levels of ubiquitinated-STAT1, pY701-STAT1 and total STAT1 were analyzed by indicated antibodies. (C) 293T cells were pretreated with MG132 (10 μM) for 1 hr, and then treated with IFNα (1,000 IU/ml) for 15 min and 30 min. pY701-STAT1 proteins were immunoprecipitated and analyzed by indicated antibodies. (D) 293T cells were pretreated with DMSO or MG132 (10 μM) for 2 hrs, and then were treated with IFNα (1,000 IU/ml) as indicated. pY701-STAT1 levels were analyzed by pY701-STAT1 antibody. (E) 293T cells transfected with Flag-STAT1 were treated with DMSO or CHX (50 μM) as indicated. Flag-STAT1 proteins were immunoprecipitated and pY701-STAT1-Flag levels were analyzed as indicated. Whole cell lysates (WCL) was utilized to determine total Flag-STAT1 levels as indicated. (F) 293T cells transfected with Flag-STAT1 were treated with CHX (50 μM) for indicated times together with DMSO or MG132 (10 μM). Flag-STAT1 proteins were immunoprecipitated, and levels of pY701-STAT1-Flag and total Flag-STAT1 were analyzed by indicated antibodies. (G) 293T cells transfected with Flag-STAT1-wild type (WT) or Flag-STAT1-K410,413R (KKRR) were stimulated with IFNα (1,000 IU/ml) for 15 min, then IFNα was removed by washing twice. Cells were further incubated as indicated times. Flag-STAT1 proteins were immunoprecipitated by Flag antibody, and levels of pY701-STAT1-Flag and total Flag-STAT1 were analyzed using indicated antibodies. (H) Quantification of pY701-STAT1-Flag protein levels from (G) (Normalized to total Flag-STAT1). (I) 293T cells preteated with MG132 (10 μM) for 2 hrs were treated with IFNα (1,000 IU/ml) for 15 min, then IFNα was removed. Cells were further incubated as indicated. pY701-STAT1 levels were detected by immunoblotting.