Figure 5.
Interaction between NKX3.1 and DYRK1B. A. 293T cells transfected with MYC-NKX3.1 and Flag-tagged mDYRK1B were lysed and subjected to immunoprecipitation with α-Flag (M2) or mouse IgG. Immunoprecipitates were analyzed by immunoblotting with either MYC or Flag antibodies. The arrows indicate specific protein bands. B.293T cells were engineered as in A to express NKX3.1 and murine DYRK1B constructs as shown below the gels. C. Dose and time-dependence of NKX3.1 association with DYRK1B. GST-DYRK1B (0-200 ng/reaction) and His-NKX3.1 were combined in an in vitro kinase assay in the presence of ATP as described in Materials and Methods. Reactions were separated on SDS-PAGE containing 25 μM Phos-tag and 50 μM MnCl2. NKX3.1 was detected by immunoblotting with NKX3.1 antiserum. D. Upper panel: 293T cells expressing MYC-NKX3.1 and transfected with either pcDNA3.1 or Flag-mDYRK1B were analyzed for phosphorylation or diluted about 200 fold with phage λ phosphatase buffer followed by concentrating with Amicon Centricon YM-10 and Amicon Ultra-0.5 ml (10K). The cleared and concentrated lysates were then treated with λ phosphatase for 60 min at 30°C and analyzed with 25 μM Phos-tag. Lower panel: 293T cells expressing MYC-NKX3.1 and transfected with either pcDNA3.1 or Flag-mDYRK1B were treated with or without 1 μM 185981 for 16 hr. NKX3.1 phosphorylation was determined by immunoblotting a gel run with 20 μM Phos-Tag.