SUMO-1 conjugation to STAT1 in vivo and in vitro. (A) Sumoylation of STAT1-WT and STAT1-KR (Lys703Arg mutant) in COS-7 cells. COS-7 cells were transfected with 2 μg STAT1-WT-HA (lanes 1-4) or STAT1-KR-HA (lanes 5-8) together with different SUMO-1 constructs (2 μg) as indicated. After 36 hours the cells were lysed in Triton X lysis buffer (50 mM Tris [tris(hydroxymethyl)aminomethane]–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 total cell lysates were immunoprecipitated with anti-HA antibodies and subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting with anti–SUMO-1 and anti-HA antibodies. (B) Sequence comparison of consensus SUMO-1 attachment sites in RanGAP1, PML, Sp100, p53, IκBα, AR, and STAT1. The consensus residues are marked in italics. The asterisk indicates the lysine that serves as a potential SUMO-1 attachment site. STAT1 sequence comparison starts at amino acid 698. (C) SUMO-1 conjugation of STAT1 in vitro. STAT1-WT (lanes 1-2) and STAT1-KR (lanes 3-4) were in vitro translated in the presence of [³⁵S]methionine using TNT-coupled transcription/translation system (Promega, Madison, WI). The in vitro–translated products were then used for an in vitro SUMO-1 conjugation reaction. The samples were resolved by SDS-PAGE gel and visualized by fluorography. (D) Sumoylation of endogenous STAT1. HeLa cells were untransfected or transfected with SUMO-1 (2 μg) plasmid using a calcium phosphate method. After 36 hours the cells were lysed as described in panel A. Sumoylation of endogenous STAT1 was analyzed after immunoprecipitation using anti-STAT1 antibody and Western blotting with anti–SUMO-1.