Fig. 2

Effect of the mutations in HeLa cells. a, b Autophosphorylation on Tyr321. Wild type (wt) and mutant GFP-DYRK1A constructs (DP and KC) were immunoprecipitated from transiently transfected HeLa cells. Phosphorylation of Y321 in the activation loop was determined by immunoblot analysis. a Illustrates the Western blots of a representative experiment. DYRK1A-Y321F (YF) was included as a control for antibody specificity. The column diagram b shows the results (means and SD) of six independent experiments. Relative tyrosine phosphorylation was calculated by relating pY321 band intensities to total protein immunoreactivity. Results are presented relative to WT. c Catalytic activity. Immunoprecipitates were subjected to radiometric kinase assays with the peptide substrate DYRKtide. In vitro kinase activities were normalized to the amount of the respective GFP-DYRK1A fusion protein as quantified by immunoblot analysis. Pairwise differences between WT and mutant DYRK1A in four independent experiments were not significant (One sample t test, p > 0.05)