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. 2010 Oct 12;29(22):3750–3761. doi: 10.1038/emboj.2010.251

Figure 1.

Figure 1

Han11 binds to HIPK2, MEKK1 and DYRK1a/b. (A) HA-tagged HIPK2 and Flag-tagged Han11 were expressed in 293T cells as shown. Equal amounts of protein contained in cell lysates were tested for adequate protein expression (input) or were used for coimmunoprecipitation experiments (IP) with the indicated antibodies. The eluted proteins were detected by immunoblotting as shown. (B) Cells expressing Flag-tagged HIPK2 with or without a hexahistidine tag were treated with the cell permeable agent DTBP to cross-link directly neighbouring proteins. After purification of His(6)-tagged HIPK2 on Ni-NTA agarose columns, the input material (INP) and the interacting endogenous Han11 protein was detected by immunoblotting. (C) Tagged versions of HIPK2 and Han11 were produced by in vitro translation. The quality of the proteins was ensured by immunoblotting of the input control (lower), direct interactions were tested by coimmunoprecipitation (upper) as shown. HA-tagged Han11 and epitope-tagged versions of the indicated kinases were produced by in vitro translation. Coimmunoprecipitation experiments were conducted to investigate the direct binding of Han11 to MEKK1 (D), DYRK1a (E) and DYRK1b (F). The interacting proteins were detected by western blotting. (G) Summary of these experiments showing direct interactions between Han11 and MEKK1, DYRK1a and DYRK1b, the C-termini of the kinases are indicated.