Sumoylation of CoCoA. (A) Schematic diagram of predicted sumoylation sites of CoCoA. AD, activation domain; LZ, leucine zipper-like sequence. (B) In vivo sumoylation of CoCoA. COS-7 cells were transfected with pSG5.HA.ARNT, pSG5.HA.CoCoA full-length, or pSG5.HA.CoCoA deletion mutants (800 ng); an EGFP-SUMO1 expression vector (200 ng) was co-transfected, where indicated. Cell extracts were collected 48 h post-transfection and analyzed by immunoblot with anti-HA antibody. Arrowheads indicate apparent sumoylation products. (C) In vitro sumoylation of CoCoA. In vitro translated 35S-labeled CoCoA(1–190) was incubated with purified sumoylation E1 (Aos1/Uba2 heterodimer) and E2 (Ubc9) enzymes in the presence of ATP/Mg2+ and SUMO1. In the mock reaction, the CoCoA was omitted. Products were analyzed by SDS-PAGE and autoradiography.