Xenbase Image ID: 117494

Figure 4. Western blot analysis of microdissected 32-cell Xenopus embryos reveals a dorso-vegetal enrichment of β-catenin. (A) Schematic diagram of how embryos were dissected into dorsal animal marginal (DAM; light gray), ventral animal marginal (VAM; black), dorsal vegetal marginal (DVM; pattern), and ventral vegetal marginal (VVM; dark gray) portions. (B) Dorsal steady-state levels of endogenous β-catenin are greater than ventral levels in untreated but not in UV-ventralized embryos. On average, the VVM portions from untreated embryos contained 77% (normalized to tubulin, three experiments) and 64% (normalized to spectrin, five experiments) as much β-catenin as the DVM zones. The VVM portions dissected from UV-irradiated embryos contained more β-catenin relative to their dorsal counterparts; 146% (normalized to tubulin, four experiments) and 172% (normalized to spectrin, four experiments). Spectrin-normalized steady-state levels of β-catenin in the VAM zone were also lower than the DAM region in untreated embryos (78%, five experiments). These differences were eliminated with UV treatment (four experiments for both tubulin and spectrin normalization). The asterisks indicate that the difference in β-catenin content between the dorsal and ventral marginal zones is statistically significant, as determined by Student's t-test (P < 0.05 for a single asterisk, P < 0.005 for a double asterisk). The error bars represent the standard error. (C) Representative Western blot detecting endogenous β-catenin, α-spectrin, and tubulin. Ventral levels of β-catenin are lower in the dissected VAM (lane 2) relative to the DAM (lane 1) regions, and in the VVM (lane 4) relative to DVM (lane 3) regions. It is worth noting that the dissected quadrants of the embryo do not precisely correspond to the areas of maximal β-catenin staining determined by confocal microscopy. Moreover, the thresholds of fluorescence chosen for Figs. 1 and 2 were set high, so as not to saturate the dorsal-ventral differences, and as a result these images do not show the detectable lower levels of cytoplasmic and membrane-associated β-catenin, which nevertheless would contribute to the signals on Western blots. Thus, one cannot quantitatively compare the dorso-ventral differences monitored by the dissections vs. confocal microscopy. Image published in: Larabell CA et al. (1997) Image reproduced on Xenbase with permission of the publisher and the copyright holder. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike license Larger Image Printer Friendly View

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