Thermal stability assay

The assay was developed based on the protocol of cellular thermal shift assay (CETSA, Jafari et al., 2014) with modifications. Stable HEK293 cells expressing wild type GFP-DYRK1A or the L295F mutant were harvested by scraping and washed with PBS at room temperature. Cells from one 10-cm dish were resuspended in 1 ml PBS supplemented with protease inhibitors. Cell suspensions were divided into 100 µl aliquots in thin-walled micro test tubes. Samples were incubated for 3 min in a thermocycler at varying temperatures as indicated and subsequently kept at 25°C for a further 3 min before snap freezing in liquid nitrogen. Different from the original method, cells were lysed with the help of a non-ionic detergent, because a significant portion of cellular DYRK1A molecules is thought to be associated with the cytoskeleton or chromatin fraction. After adding Triton X-100 to a final concentration of 0.1%, cells were incubated on ice for 15 min before soluble fractions were separated from precipitated proteins by centrifugation (20 min, 20,000 g at 4°C). The abundance of DYRK1A in the soluble fraction was quantified by western blotting.