Fig. 6

Equivalent levels of Myc-tagged protein are made in embryos injected with RNAs encoding wild-type and homeodomain- deficient myc-zOtx1 RNAs. An SDS–PAGE gel on which was run extract from the equivalent of two embryos injected with myc-zOtx1-wt RNA (WT) or myc-zOtx1-ΔH RNA (ΔH), or that were uninjected (C), was Western blotted as described under Materials and Methods. Bands indicated with an asterisk are endogenous proteins recognized by the anti-mouse Ig second antibody and serve as a loading control. The position of the myczOtx- wt protein is indicated by a black arrowhead, that of the myc-zOtx1-ΔH protein by an open arrowhead. The protein made by embryos injected with myc-zOtx1-wt or myc-zOtx1-ΔH RNAs each ran with mobilities 21 kDa higher than expected, as did proteins made from these RNAs by coupled in vitro transcription and translation (using the TnT coupled reticulocyte lysate system; Promega), indicating a consistent anomalous mobility of myczOtx1 proteins in the SDS–PAGE system. In this experiment, myc-zOtx1-wt and myc-zOtx1-ΔH RNAs that had been used for the injections in Table 1 were also each injected at the 1- to 8-cell stage and samples were prepared from these embryos at the 100% epiboly stage for immunoblotting. Similar equivalence has been shown for embryos injected into 1 cell at the 16-cell stage and allowed to develop to 14 hpf.