Fig. 4. SOX3 mutant activities. (A) HeLa cells were transfected with plasmids encoding the various XSOX3 mutant polypeptides, along with a plasmids encoding a mutationally stabilized form of Xenopus ß-catenin, the pRL-TK plasmid for the normalization of transfection efficiency and the TOPFLASH reporter. Co-expression of ß-catenin (CAT +) activated TOPFLASH and this activation was suppressed by the co-expression of XSOX3-V5H₆ (WT). The co-expression of the mutant XSOX3 polypeptides (m68 was not tested) led to the inhibition of the ß-catenin-induced activation of TOPFLASH. (B) Embryos were injected equatorially into the two dorsal blastomeres of four-cell embryos with RNA encoding V5H₆-tagged forms of XSOX3. At stage 12, embryos were homogenized and the lysates immunoprecipitated using the mouse antiV5 antibody and then analyzed by SDS-PAGE/immunoblot using the mouse antiV5 antibody. Two distinct experiments are displayed. In each case, similar amounts of the exogenous polypeptides were found to accumulate. No signal was detected in uninjected (Un) embryos. (C) Control and RNA-injected embryos were allowed to grow out to stage 25. A wild-type (DAI 5) embryo is show at the top of the panel; three embryos displaying various levels of ventralization are shown below. (D) The proportion of embryos ventralized by the injection of XSOX3-V5H₆ RNA or its mutated variants is displayed. The exact numbers and extent of ventralization observed are given in Table 2. Wild-type embryos are 0% ventralized.