Fig. 8

LZIC and β-catenin do not interact. (A) LZIC and β-catenin do not interact in vitro. ³⁵S-labeled proteins were tested for their ability to interact. The immunoprecipitated samples are shown above the lane numbers, and the input samples are shown below the lane numbers. HA-tagged β-catenin (β-cat-HA) was incubated with mouse ICAT (mICAT), mouse LZIC (mLZIC), the ICAT homology domain (IHD) of mouse LZIC (mIHD), or the IHD of zLZIC (zIHD), as indicated in the figure. Only ICAT was co-precipitated by β-cat-HA (lane 6). (B) LZIC and β-catenin do not interact in vivo. Xenopus embryos were injected with RNA encoding a FLAG-tagged β-catenin (βcat-FLAG) and either HA-tagged Tcf3 (Tcf3-HA), zebrafish LZIC (HA-LZIC), or the zebrafish IHD (HA-IHD), alone or in combination as indicated in the figure. After 4 h, translated protein was isolated from the embryos, and βcat-FLAG (middle panel) or HA-tagged proteins (right panel) were immunoprecipitated to test for the ability of co-expressed proteins to co-precipitate. Total lysate was also analyzed to ensure that all of the proteins were expressed in the embryo (left panel). Only Tcf3-HA co-precipitated with βcat-FLAG (middle panel, lane 6). Similarly, βcat-FLAG could only be co-precipitated by Tcf3-HA (right panel, lane 6). Western blot: anti-HA/anti-FLAG. (C) The armadillo repeat region of β-catenin does not interact with LZIC. Autoradiograph of ³⁵S-labeled proteins produced in vitro. The armadillo repeat region of β-catenin (GST-Arm) was tested for its ability to interact with ICAT, mIHD, zIHD, and zLZIC, as indicated in the figure. Input proteins are shown in lanes 1–5. Only ICAT was able to interact with GST-Arm (lane 11).