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. 2000 Dec 19;97(26):14394–14399. doi: 10.1073/pnas.97.26.14394

Figure 4.

Figure 4

Ski interacts with Smad1 and Smad5. (A) Flag-tagged full-length or truncated Smad proteins were cotransfected into 293T cells together with HA-tagged Ski. The Smad-bound Ski was isolated by immunoprecipitation with anti-Flag M2 mAb and detected by Western blotting with an anti-HA mAb. Cell lysates were blotted directly as a control for HA-ski expression. (B) Ski associated with endogenous Smad1 and Smad4 in W-20-17 cells. W-20-17 cells were transfected with Flag-Ski together with or without the constitutively active type I BMP receptor. Ski-associated Smad complex was isolated by immunoprecipitation with anti-Flag agarose and detected by immunoblotting with an anti-Smad1 (T-20) or anti-Smad4 (C-20) antibody. (C) Ski binds to Smad1 directly in vitro. GST-Ski immobilized on glutathione-Sepharose was incubated with purified recombinant Smad1 or Smad4 as described in Materials and Methods. The Ski-bound Smads were eluted by glutathione and detected by immunoblotting with anti-Smad1 or anti-Smad4. GST alone was used as negative control. The amounts of wild-type or mutant GST-Ski used in the binding assay were shown by Coomassie staining. * indicates nonspecific background bands.