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. 2010 Mar 12;285(19):14415–14423. doi: 10.1074/jbc.M109.074583

FIGURE 2.

FIGURE 2.

PIASy stimulates PARP1 modification by SUMO-2/3 in reconstituted in vitro SUMOylation reaction. All recombinant proteins, E1, E2, E3, PARP1, and SUMO, were expressed in E. coli and purified as described under “Experimental Procedures.” In vitro SUMOylation reaction was performed with 15 nm E1, 500 nm PARP1, and 6 μm SUMO at 25 °C. Reactions were terminated at the indicated times by the addition of SDS-PAGE sample buffer. SUMOylation of PARP1 was analyzed by immunoblotting with horseradish peroxidase-conjugated anti-T7 antibody (Novagen). The bracket indicates the position of SUMOylated PARP1. A, Ubc9 dose dependence of PARP1 SUMOylation without E3 is shown. B, kinetics of Ubc9-dependent PARP1 SUMOylation are shown. The SUMOylation reaction was performed with the indicated concentration of Ubc9 without PIASy. The reaction mixtures were incubated for 60 min in A and for the indicated time periods in B. C, PIASy-dependent SUMOylation of PARP1 is shown. The reaction mixtures containing the indicated concentration of Ubc9 and PIASy were incubated for the indicated times.