Western Blot quantification of DYRK1A protein

Isolated protein lysates (20 μg) from the cerebral cortex, hippocampus, and cerebellum were resolved by electrophoresis on polyacrylamide gels (Bolt 4–12% Bis Tris Plus Gels), then transferred to PVDF membranes. Membranes were blocked in 5% milk in Tris Buffered Saline with 0.1% Tween 20 (TBS-T), incubated overnight at 4 °C in primary antibodies diluted in 5% milk-TBS-T as follows: rabbit anti-DYRK1A antibody, 1:500 (A303–802A, Bethyl Laboratories); mouse anti-beta-actin, 1:5000 (A2228, Sigma Aldrich), and labeled with donkey anti-rabbit IgG AlexaFluor 790 and donkey anti-mouse IgG AlexaFluor 680 secondary antibodies (1:10,000, Jackson Immunoresearch). Fluorescence was detected using a LI-COR CLx Imager. Each membrane included protein samples from each of the three brain regions from four different mice, one from each genotype and treatment combination. DYRK1A for each sample was normalized to actin and each normalized ratio was expressed as a proportion relative to the mean of the euploid-PBS group for the particular brain region.