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ig. S4. Immunoprecipitation of flag-xTGIF2 with both Smad1 and Smad2. Flag-tagged TGIF2 or Myc-tagged FAST1 were injected into vegetal blastomeres (dorsally for Smad2 IP; ventrally for Smad1 IP) of four-cell stage embryos. Embryonic lysates were immunoprecipitated with anti-Smad1 or anti-Smad2 and analyzed by immunoblot with anti-flag and anti-myc antibodies. Myc-Fast1 was used as control for the specificity of the immunoprecipitation reactions, for its ability to interact exclusively with Smad2 (Chen et al., 1997). Flag-xTGIF2 immunoprecipitated complexes are indicated with **, and the myc-Fast1 complex with *. A portion of the lysates was blotted with anti-flag and anti-myc directly (without immunoprecipitation) to assess the expression of flag-xTGIF2 and myc-Fast1, respectively. In addition, equal expression of Smad1 and Smad2 was tested by immunoblotting on the crude extracts using anti-Smad1 and anti-Smad1 antibodies. TrueBlot IP beads and HRP-conjugated secondary antibodies were used to minimize interference by the heavy and light chains of the immunoprecipitating antibody in the IP/IB experiments. Chen, X., Weisberg, E., Fridmacher, V., Watanabe, M., Naco, G. and Whitman, M. (1997). Smad4 and FAST-1 in the assembly of activin-responsive factor. Nature389, 85-89. Image published in: Spagnoli FM and Brivanlou AH (2008) Copyright © 2008. Image reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License. Larger Image Printer Friendly View

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