In vitro kinase activity assay

In vitro kinase assays were carried out in a total final volume of 30 μL in a kinase buffer containing 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10 mM MgCl₂, and 1 mM DTT. Reactions including 1 μg of Adi3 and 3 μg of each substrate were started with the addition of 1 μCi of [γ-³²P]ATP (6,000 Ci/mmol, Perkin-Elmer) and non-radiolabeled ATP to a final concentration of 20 μM per reaction followed by incubation for 1 hour at RT. Reactions were terminated by the addition of 10 μL 4X SDS-PAGE sample buffer and separated by 8% SDS-PAGE. The proteins in the gels were visualized using GelCode Blue Stain Reagent (Thermo Fisher Scientific), and gels were dried and exposed overnight to a phosphor screen. Visualization and quantification of incorporated radioactivity were conducted using a phosphorimager (Typhoon FLA7000, GE Healthcare Life Sciences) and quantification software (ImageQuant TL, GE Healthcare Life Sciences).

The Adi3 kinase assays with Gal83 substrate (see S1A Fig) were initially done in our previous study [28]. These assays were repeated here because the kinase assay conditions in the current study have changed since our previous study. In the previous study [28], 0.4 μg of Adi3, 2 μg of Gal83, and 0.25 μCi of [γ-³²P]ATP were used and reactions were incubated for 30 min. All of these amounts and the reaction time were increased in the current study as described above. While there were differences in Gal83 absolute phosphorylation levels between the two sets of data, the trends are the same: Adi3^S212D/S539D showed the highest trans-phosphorylation on Gal83 in both experiments.