Substrate quality-determining amino acid residues of AtSUMO3-ScPCNA conjugates. ScPCNA was conjugated in vitro with either AtSUMO3 wild-type protein (A; lane CA), the AtSUMO3 variant M90/Q (B; lane CB), or the AtSUMO3 triple mutant A89/H + M90/Q + S91/T (C; lane CC). Conjugates were treated with either ScULP1 (lanes 2), ESD4 (lanes 3), At1g10570 (lanes 4), At1g60220 (lanes 5), or XopD (lanes 6). Proteins were separated on a 12% SDS-polyacrylamide gel, and cleavage of AtSUMO3-ScPCNA conjugates was detected by western-blot analysis using a Myc peptide-specific antiserum.