Figure 2.

Axin interacts with Smad7, and Axin, Arkadia, and Smad7 form a ternary complex. (A) Identification of Smad7 as another novel Axin-interacting protein. FLAG-tagged Smads 1–7 were separately cotransfected with HA-Axin into 293T cells. Reciprocal immunoprecipitation with anti-FLAG and anti-HA was carried out, followed by Western blotting analysis. Smad3, 6, and 7 (marked by asterisks) were co-precipitated with Axin. (B) Endogenous Axin and Smad7 interact with each other in 293T cells. The 293T cell lysate was incubated with goat Smad7 polyclonal antibody (Santa Cruz Biotech.), followed by Western blotting with rabbit AxinC2b polyclonal antibody. Three separate experiments were carried out and similar results were obtained. (C) Two-step co-immunoprecipitation to test for ternary complex formation of Axin, Arkadia, and Smad7. The procedures of two-step co-immunoprecipitation are outlined in the left box. HEK293T cells were transfected with Myc-Smad7, FLAG-ArC937A, and HA-Axin (or untagged Axin as control, marked by asterisk). The first immunoprecipitation was performed with anti-HA antibody. The complex was eluted by using HA peptide, followed by the second step of immunoprecipitation with anti-Myc or control mouse IgG. Protein samples from each step were then subjected to Western blotting analysis separately by using anti-Axin, anti-Myc, and anti-Arkadia antibodies. The experiment was repeated with essentially the same result. (D) Axin increases the interaction affinity of Arkadia for Smad7. FLAG-ArC937A and Myc-Smad7 were cotransfected with or without Axin into 293T cells. Immunoprecipitation was carried out with anti-FLAG, followed by immunoblotting with anti-Myc to detect Smad7, and anti-HA to detect Axin. Increased amount of Myc-Smad7 was co-immunoprecipitated with Arkadia from cells coexpressing Axin.