In vitro kinase assays

For the IVK assays, immunocomplexes or GST-fusion proteins expressed in bacteria (see Supplementary Methods) were incubated for 20 min at 30 °C, in the presence or absence of GST-DYRK1A, in 20 µl of phosphorylation buffer (25 mM Hepes [pH 7.4], 5 mM MgCl₂, 5 mM MnCl₂, 0.5 mM DTT) that contained 50 µM ATP and 2.5 µCi [γ-³²P]-ATP (3,000 Ci/mmol, Amersham Biosciences). The incorporation of ³²P was determined by SDS-PAGE and exposing the dried gel to film.

DYRK1A kinase activity was assessed using the DYRKtide peptide as the substrate⁴⁰. Briefly, DYRK1A-immunocomplexes were incubated for 20 min at 30 °C in 20 µl of phosphorylation buffer including 200 µM DYRKtide and 2.5 µCi [γ-³²P]-ATP (3,000 Ci/mmol). Aliquots of the reaction were dotted onto P81 Whatman phosphocellulose paper in triplicate, washed extensively with 5% orthophosphoric acid and counted in a liquid scintillation counter (Beckman Coulter).