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Figure 3. Immunolocalization of proteins unrelated to β-catenin. To assess whether the patterns of β-catenin observed in the eight-cell embryo (Fig. 1) were unique or were shared by other proteins, we undertook a comparable analysis of eight-cell embryos stained with other antibodies, optically sectioned near the equator, and viewed at low magnification to monitor dorsoventral patterns (A–C) or viewed at the membrane at high magnification (D–F). Dorsal is to the right for A, B, and C as in Fig. 1. While eight-cell embryos show a clear dorsal enrichment of β-catenin (Fig. 1, A and B), membrane skeleton protein 4.1 (A), α-spectrin (B), and cytokeratin (C) antibodies show no dorsal enrichment. The α-spectrin image in B is somewhat more pigmented in the animal hemisphere than the other images, hence the ventral blastomeres appear somewhat darker than the dorsal blastomeres (right) owing to pigmentation differences. The cytokeratin image is yellow because of the extensive overlap of the green autofluorescence signal with the red cytoplasmic staining of the antibody (see F for cytoplasmic stain at higher magnification). While β-catenin staining extends extensively from the membrane through the cytoplasm at the eight-cell stage (Fig. 1, A and B), both protein 4.1 (D) and α-spectrin (E) are restricted to the plasma membrane, which is consistent with their known functions in the membrane skeleton, while the cytokeratin staining extends into the cytoplasm (F) as filaments as previously noted (Klymkowsky et al., 1987), though our fixation methods also retain nonpolymeric protein and increase nonfilamentous cytokeratin staining. |
