E. coli-based in vitro translation assay

The PURExpress kit (New England Biolabs) was employed. DYRK1A was expressed at 37 °C for 40 min in a 12.5-μl reaction mixture containing 30 ng of pET28a(+)-FLAG-DYRK1A. For inhibitor experiments, FINDY or RD0392 was added prior to the initiation of transcription. For immunoprecipitation, 100 μl of PBS containing 0.5% Empigen BB, 0.5 mg ml⁻¹ BSA and 5 mM EDTA was added into the reaction mixture, then incubated with anti-FLAG (M2) beads at 4 °C for 6 h. The samples were prepared with an SDS-urea buffer, then analysed with SDS–PAGE followed by western blot (Supplementary Fig. 14). The band intensities were measured using the LAS-3000 (Fujifilm) and Multi Gauge software (Fujifilm).

For the phosphatase experiment, immunoprecipitated FLAG-DYRK1A bound on the beads was incubated in a total volume of 100 μl containing 1,600 U of lambda protein phosphatase for 2 h at 30 °C. After washing the beads with autophosphorylation reaction (AR) buffer (see below) containing 0.1 mg ml⁻¹ of BSA (Sigma-Aldrich), the beads were incubated with the indicated concentrations of ATP and sodium orthovanadate (1 mM) for 2 h at 37 °C and prepared with the SDS-urea buffer. To check the residual phosphatase activity, non-treated immunoprecipitated FLAG-DYRK1A bound on the beads was added into the reaction.

For the in vitro kinase assay, 0.5 μl of the reaction mixture was incubated in a total volume of 25 μl of AR buffer containing 0.1 mg ml⁻¹ of BSA, 100 μM of ATP, 50 μM of DYRKtide peptide (Anaspec) and the indicated concentrations of the small molecules for 2 h at 37 °C. The consumption of ATP was measured with ADP-Glo Kinase Assay kit (Promega Corporation).