![Figure 1. Mutation of the SUMO attachment site modulates STAT1-mediated transcription. (A) Point mutations in the SUMO consensus site affect SUMO-1 conjugation to STAT1. COS-7 cells were transfected with 0.7 μg of STAT1-E705A-HA, STAT1-I702R-HA, and STAT1-WT-HA together with SUMO-1 plasmid (0.2 μg) as indicated. After 36 hours the cells were lysed in Triton-X lysis buffer (50 mM tris(hydroxymethyl)aminomethane–HCl [Tris-HCl], pH 7.4; 150 mM NaCl; 1 mM EDTA [ethylenediaminetetraacetic acid]; 50 mM NaF; 5 mM NEM; 1% Triton-X 100; and 10% glycerol and protease inhibitors) and equal amounts of lysates were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with anti–SUMO-1 and anti-HA antibodies. (B) K703R and E705A mutations increase the transcription activity of STAT1. STAT1-WT-HA, STAT1-K703R-HA, and STAT1-E705A-HA were transfected in U3A cells together with GAS–luciferase (luc) reporter plasmid (0.5 μg) and plasmid cytomegalovirus–βgalactosidase (pCMV-βGAL; 0.25 μg). After 24 hours the cells were starved and stimulated with 10 ng/mL huIFN-γ for 6 hours. The ± SD were calculated as a mean of 2 independent experiments. The bottom panel shows the STAT1 protein levels analyzed by Western blotting using anti-HA antibodies. RLU indicates relative luciferase units. (C) K703R mutation enhances DNA binding of STAT1. U3A STAT1-WT and STAT1-KR stable clones were treated with IFN-γ (100 ng/mL) as indicated and STAT1 DNA binding was analyzed by EMSA with 32P-labeled GAS-IRF1 probe. The bottom panel indicates the STAT1 protein levels by anti-HA immunoblotting. (D) Analysis of GBP1, TAP1, and IRF1 gene activation in U3A STAT1-WT and STAT1-KR stable clones. Total RNA isolated from U3A stable clones stimulated with IFN-γ (10 ng/mL) for different times as indicated was subjected to RT-PCR using specific primers for GBP1, TAP1, and IRF1. The target gene expression levels were normalized against the expression values of GAPDH3. The results were calculated as the mean ± SD of 2 independent experiments for each type of clone.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/106/1/10.1182_blood-2004-11-4514/4/zh80130580400001.jpeg?Expires=1724232244&Signature=3U0cY~~WsNhz7nMWVLZMIblhoZPwQ8iXzTnaMalIto1c8LJQFYPEasQH30Dwl3tds-UAuqwQ2LXyx8hGG-mcxzDkxrDsJuv3cNhZfk459Op19BSpjut7ibid2CpOdEhAZdJfnXNoSifO8h~SQxsawyd0Yjz18KTxy~De7DIo4KfzNv6-XzZ3cX4fXyW859Y6i1KP0mnJlKxz600MZWzT7XfshYokC0pHBMSU21CmftN~H29GlEJuzdYAoqVNUXliQgd9CIf8k553VPXX2PNgVF0pfGDJnR9hquRbr~1r9IU1jFL7bURMmwwkZ8U0AjYha9PHKeAvG9ospx5GJlbOvw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Mutation of the SUMO attachment site modulates STAT1-mediated transcription. (A) Point mutations in the SUMO consensus site affect SUMO-1 conjugation to STAT1. COS-7 cells were transfected with 0.7 μg of STAT1-E705A-HA, STAT1-I702R-HA, and STAT1-WT-HA together with SUMO-1 plasmid (0.2 μg) as indicated. After 36 hours the cells were lysed in Triton-X lysis buffer (50 mM tris(hydroxymethyl)aminomethane–HCl [Tris-HCl], pH 7.4; 150 mM NaCl; 1 mM EDTA [ethylenediaminetetraacetic acid]; 50 mM NaF; 5 mM NEM; 1% Triton-X 100; and 10% glycerol and protease inhibitors) and equal amounts of lysates were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with anti–SUMO-1 and anti-HA antibodies. (B) K703R and E705A mutations increase the transcription activity of STAT1. STAT1-WT-HA, STAT1-K703R-HA, and STAT1-E705A-HA were transfected in U3A cells together with GAS–luciferase (luc) reporter plasmid (0.5 μg) and plasmid cytomegalovirus–βgalactosidase (pCMV-βGAL; 0.25 μg). After 24 hours the cells were starved and stimulated with 10 ng/mL huIFN-γ for 6 hours. The ± SD were calculated as a mean of 2 independent experiments. The bottom panel shows the STAT1 protein levels analyzed by Western blotting using anti-HA antibodies. RLU indicates relative luciferase units. (C) K703R mutation enhances DNA binding of STAT1. U3A STAT1-WT and STAT1-KR stable clones were treated with IFN-γ (100 ng/mL) as indicated and STAT1 DNA binding was analyzed by EMSA with 32P-labeled GAS-IRF1 probe. The bottom panel indicates the STAT1 protein levels by anti-HA immunoblotting. (D) Analysis of GBP1, TAP1, and IRF1 gene activation in U3A STAT1-WT and STAT1-KR stable clones. Total RNA isolated from U3A stable clones stimulated with IFN-γ (10 ng/mL) for different times as indicated was subjected to RT-PCR using specific primers for GBP1, TAP1, and IRF1. The target gene expression levels were normalized against the expression values of GAPDH3. The results were calculated as the mean ± SD of 2 independent experiments for each type of clone.
