Appendix 1—figure 1. Reactions were assembled in two separate mixtures: an E1/E2 mix that contained excess unlabeled Cyclin E peptide (tube 1) and an SCF-³²P-labeled substrate mix (1 μM SCF, 0.2 μM substrate; tube 2).

Following addition of 10 μM E2 and/or 2.5 μM ARIH1 to tube one already containing reaction buffer (30 mM Tris-HCl (pH 7.5), 100 mM NaCl, 5 mM MgCl₂, 2 mM DTT and 2 mM ATP), E1 (0.5 μM) and ubiquitin (80 μM), each mix was incubated at room temperature for at least 8 min. The E1/E2 mix was then pipetted into the SCF/substrate mix initiating the reaction, which was then briefly vortexed and quenched in reducing 2x loading SDS-PAGE buffer after 10 s. The reactions were resolved by SDS-PAGE and visualized by autoradiography. All reactions were performed in duplicate.