In vitro SUMO conjugation assay. (A) Purified recombinant Stat1 (0.5 μM) is SUMO-ylated in vitro after incubation with SUMO-1 (2 μM), E1 (Uba2/Aos1; 0.3 μM), E2 (Ubc9; 0.3 μM), and E3 (PIAS1 or Nup358 at 0.3 μM) for 1 hour at 37°C. Samples were fractionated by SDS-PAGE and immunoblotted with anti-Stat1. Mobility of molecular weight markers and Stat1 isoforms are indicated. (B) Wild-type and mutant p53, Stat1, PO4-Stat1, and Stat3 peptides (500 μM) were SUMO-ylated, as in panel A, in the absence of E3, with either wild-type or mutant Ubc9 for 5 or 24 hours at 37°C. Samples were fractionated by SDS-PAGE and stained with Coomassie Blue. (C) Graphic representation of a more detailed kinetic SUMO-ylation assay of peptides from panel B (t = 0, 10, 40, 60, 90, 160, and 300 minutes). Maximal conjugation is 1.0. Products were detected by staining the gel with Sypro (Bio-Rad). The images were quantified on Quantity One Software (Bio-Rad). Error bars indicate standard deviation.
