Figure 1.

NLK and HIPK2 bind to the DBD of A-Myb. (A) GST pulldown assay. (Top) The functional domains of A-Myb and the deletion mutants used are shown schematically. The results of binding assays shown below are indicated on the right. (Bottom) The input lanes show the ³⁵S-A-Myb variants, which were translated in vitro. The ³⁵S-A-Myb variant indicated above each lane was mixed with GST-NLK or GST-HIPK2C affinity resin, which have been described previously (Kanei-Ishii et al., 2004a), and the bound proteins analyzed by SDS-PAGE and autoradiography. (B) Coimmunoprecipitation of A-Myb and NLK. Lysates were prepared from CV-1 cells transfected with a mixture of FLAG-A-Myb and HA-NLK expression plasmids. Lysates were precipitated by anti-HA antibody or control IgG and the immunocomplexes were analyzed by Western blotting using anti-FLAG or anti-HA antibodies. HA-NLK and FLAG-A-Myb in the whole cell extracts were detected by Western blotting (lower two panels). (C) Binding of B-Myb to NLK and HIPK2. In vitro-translated ³⁵S-B-Myb was mixed with the GST-NLK or GST-HIPK2C affinity resin, and the bound proteins were analyzed by SDS-PAGE and autoradiography.