In vitro sumoylation

APC4 wild-type, APC4^K772A, APC4^K798A, APC4^K772/798A, and RanGAP1 were produced by in vitro transcription and translation in rabbit reticulocyte lysate in the presence of [³⁵S]methionine (Perkin Elmer, Waltham, MA) using the TNT T7 Quick Coupled Transcription Translation Kit according to the manufacturer’s instructions (Promega, Madison, WI). 2 μL of translation product was added to a 30 μL reaction containing 200 nM human SUMO E1 enzyme, 600 nM human Ubc9, 1.0 μM human SUMO-1 or SUMO-2 proteins, 1 mM ATP, 20 units/mL creatine phosphokinase, 5 mM phosphocreatine, 0.6 mg/ml inorganic pyrophosphatase from E. coli, 20 mM HEPES-KOH (pH 7.3), 110 mM potassium acetate, 2 mM magnesium acetate, and 1 mM dithiothreitol (DTT). Reactions were incubated at 30°C for the indicated times and stopped by addition of 2x SDS-sample buffer and analyzed by SDS-PAGE followed by autoradiography. In vitro sumoylation assays are described in more detail at Bio-protocol (Lee et al., 2018).

Recombinant SUMO proteins and SUMO enzymes were purified from E. coli as previously described (Yunus and Lima, 2009).