PIAS proteins and activation of STAT1 promote SUMO-1 conjugation. (A) PIAS proteins promote SUMO-1 conjugation to STAT1-WT in vivo. COS-7 cells were transiently transfected with plasmids encoding STAT1-WT-HA (2 μg) and SUMO-1 (0.6 μg) together with Flag-PIAS1 (2.5 μg), Flag-PIAS1mut (1 μg), PIAS3 (4 μg), ARIP3 (4 μg), and ARIP3mut (0.5 μg) as indicated. The plasmid amounts were adjusted to yield similar expression levels. After 36 hours the cells were lysed in Triton X lysis buffer, and STAT1 protein was immunoprecipitated using anti-HA antibody and analyzed by immunoblotting as indicated. PIAS and PIASmut protein levels were analyzed by immunoblotting of 15 μg total cell lysates with anti-Flag antibody. (B) IFN-γ stimulation enhances SUMO-1 conjugation to STAT1. COS-7 cells were transfected with plasmids encoding STAT1-WT-HA (2 μg) and SUMO-1 (1 μg). After 36 hours the cells were serum starved and stimulated with 100 ng/mL human IFN-γ for different time points as indicated. STAT1 was immunoprecipitated and analyzed by immunoblotting. (C) Pervanadate-induced phosphorylation of STAT1 enhances SUMO-1 conjugation. HeLa cells were starved overnight and left unstimulated or stimulated with 50 μM pervanadate for 30 minutes. Total cell lysates were prepared as described in Figure 1A, and STAT1 was immunoprecipitated with anti-STAT1 antibody, and immunoblotting was performed with anti–SUMO-1 or anti-STAT1 antibody. (D) The effect of Lys703Arg mutation on STAT1-mediated gene activation in response to IFN-γ stimulation. U3A clones stably expressing STAT1-WT-HA (U3A-WT1 and U3A-WT2) or STAT1-KR-HA (U3A-KR1 and U3A-HA-KR2) were transiently transfected with SUMO-1 (1 μg), GAS-luc reporter plasmid (0.5 μg), and pCMV-βGal. After 24 hours the cells were starved in 0.5% serum and left unstimulated or stimulated with 10 ng/mL human IFN-γ for 6 hours followed by luciferase measurement. The mean relative luciferase units ± SD from 3 independent experiments are shown. The lower panel shows the STAT1 levels in different clones.