Figure 3.
DYRK1a/b and HIPK2 interact directly in the absence of Han11. (A) 293T cells were transfected with Han11-specific shRNA (shHan11) or with a control shRNA construct (shControl), which differs in two nucleotides from shHan11. One day after transfection, the cells were selected with puromycin for another 24 h. Then, the cells were retransfected to express Myc-DYRK1a, Flag-HIPK2 and the vectors directing the synthesis of the indicated shRNAs for another 30 h as indicated. Equal amounts of proteins were immunoprecipitated with anti-Flag or isotype-matched IgG control antibodies. Immunoblotting was used to detect the (co)immunoprecipitated proteins, 10% of the material was used for the input (INP) control. (B) The experiment was performed as in (A) with the exception that the interaction between expressed HIPK2 and DYRK1b was analysed. (C) Flag-tagged HIPK2 was coexpressed with GFP-tagged DYRK1a or DYRK1b in U2OS cells. HIPK2 was visualized by staining with anti-Flag and anti-Cy3 antibodies, whereas the nuclei were stained with Hoechst. (D) Model of the direct, Han11-independent interaction between HIPK2 and both DYRK1 isoforms.