English: (A) 293T cells in six-well plates were cotransfected with plasmids expressing IE1 (1 µg), flag-SUMO-1 (1 µg), and increasing amounts of SRT-PIAS1 (0.3, 1, and 3 µg), as indicated. At 48 h, total cell lysates were prepared and immunoblotted with an anti-IE1 antibody. (B) 293T cells were cotransfected with plasmids expressing IE1 (1 µg), flag-SUMO-1 (1 µg), and wild-type or C351S mutant SRT-PIAS1 (0.5 µg), as indicated. At 48 h, total cell lysates were prepared and immunoblotted with anti-IE1 or anti-SRT antibodies. (C) In vitro SUMOylation reactions were conducted with bacterially purified His-IE1, GST-SAE2/1, His-Ubc9, and GST-SUMO-1GG proteins, and immunoprecipitated flag-PIAS1 proteins (see Materials and Methods). The reaction products were analyzed by SDS-PAGE (8%) and immunoblot assays with anti-IE1 antibody. The amounts of flag-PIAS1 protein used were also shown by immunoblotting with anti-flag antibody.