Fig. 5. DYRK1A accumulation in nuclear speckles is not dependent on its kinase activity. (A) COS-7 cells were transfected with wild-type HA-DYRK1A, the mutant HA-DYRK1A K179R and the empty vector as a control. HA-tagged proteins were immunoprecipitated with an anti-HA antibody, and the immunocomplexes subjected to an in vitro kinase assay using the synthetic peptide DYRKtide as an exogenous substrate. A western blot for both inputs (i) and immunoprecipitates (IP) was developed with an anti-HA antibody to control for equal presence of the fusion proteins (left panel). Autophosphorylation of DYRK1A was detected by resolving the kinase reactions in 10% SDS-PAGE and autoradiography of the dried gels (middle panel). Incorporation of ³²P into DYRKtide was determined in triplicate by dotting aliquots of the reaction onto phosphocellulose paper. The average count is shown on the chart (right panel). (B) COS-7 cells were transfected with GFP-fusion proteins of DYRK1A wt (upper panel) and the kinase-negative mutant described above (DYRK1A K179R) (lower panel) and co-stained with the antibody anti-SC35 to detect the colocalization of DYRK1A and SC35 in nuclear speckles.