FIGURE 3.

PICH^hs is SUMOylated in vitro in a PIASy-dependent manner, and PICH^hs SUMOylation reduces DNA binding in vitro. a and b, in vitro SUMOylation reactions were performed as described previously (12, 14) with 40 nm E1, 80 nm Ubc9, 40 nm PIASy, 0.5 μm PICH^hs or PAPR1, and 10 μm SUMO2, unless otherwise indicated, and were analyzed by immunoblotting using an anti-T7 antibody conjugated to HRP. PIASy is required for robust PICH SUMOylation. SUMOylation reactions with increasing concentrations of Ubc9 in the absence of PIASy (a) and the reaction with increasing PIASy are shown (b). Brackets indicate SUMOylated species. c, effect of PICH SUMOylation on PICH/DNA interaction. PICH^hs was SUMOylated (SPICH^hs) in vitro and incubated with linear pBluescript plasmid. PICH^hs-DNA interactions were observed using agarose gels, followed by EtBr staining. The bracket indicates the non-shifted DNA position. d, quantification of the percentage of non-shifted DNA in the EMSA. The p values and S.D. were calculated from three independent experiments.