Plasmids. Plasmids expressing GST-SAE2/SAE1 and His-Ubc9 have been described previously [29]. A pET-17b (Novagen)-based plasmid expressing T7-SUMO-1GG was kindly provided by Ho Zoon Chae (Chonnam National University, Republic of Korea). Plasmids expressing flag-SUMO-1 and HA-IE2 were generated in a pSG5 background [30] and
Staurosporine plasmid for myc-IE2 was generated on a background of pCS3-MT using Gateway technology (Invitrogen). Plasmids for GST-IE1, GST-PML VI, GST-IE2(135–289), and GST-Sp100A were generated in a pGEX-derived background also using Gateway technology [31]. Plasmid expressing GST-RanGAP1(NΔ419) has been previously described [11]. To generate plasmid expressing IE2 SIM mutant, the IVIS residues between 200 and 203 were replaced with A residues using the Stratagene QuickChange site-directed mutagenesis protocol. Ubc9 K14R mutant was generated using the same mutagenesis protocol. His-tagged SUMO-1-Ubc9(K14R) fusion was generated in a pDEST17 (Invitrogen) background by placing Ubc9 behind SUMO-1ΔGG, a truncated SUMO-1 without the six carboxyl-terminal
amino acids including double-glycine residues. Plasmids for GFP-Ubc9 and GFP-SUMO-Ubc9 fusion constructs were also generated in a pDEST53 (Invitrogen) background by placing Ubc9 or SUMO-Ubc9 behind GFP using Gateway technology.
