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Fig. 4. Effects of overexpressing aPKC constructs on expression of superficial and deep layer markers. Whole-mounts and sections of st.16 Xenopus embryos showing in situ hybridisation for (A) the superficial ectodermal marker Uroplakin 1B and (B) the deep ectodermal marker Prothymosin alpha. The dotted line marks the boundary between ectoderm (above) and mesendoderm (below). Both ectodermal layers are thickened following injection of either aPKC-CAAX or NLS-aPKC-CT mRNA. n, total number for embryos analysed. Scale bars: 250 μm. |
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Fig. 2. Subcellular localisation and expression of aPKC constructs. (A) Immunohistochemistry using anti-aPKC antibody (green) on sections showing ectoderm of st.10.5 Xenopus embryos (apical side up) from non-injected control (left) or injection with aPKC-CAAX or NLS-aPKC-CT mRNAs. Arrows indicate nuclear staining. (B) Immunocytochemistry on HeLa cells transfected with the constructs shown and assayed with anti-aPKC (red) and anti-GFP (green) antibodies. Nuclei are blue (DAPI staining). (C) Western blot analysis with nuclear lysates from HeLa cells transfected with HA-tagged constructs as shown and assayed with anti-aPKC and anti-HA-antibodies. Endogenous aPKC shows some nuclear localisation, as shown by the anti-aPKC antibody, but HA-tagged aPKC-CAAX shows greater nuclear accumulation than HA-aPKC, as shown by the anti-HA antibody. P95 and tubulin were used as nuclear and cytoplasmic controls, respectively. (D) Western blot analysis with nuclear lysates from st.10.5 Xenopus embryos injected with HA-tagged constructs as shown and blotted with anti-HA and anti-aPKC antibodies. Both HA-tagged aPKC and aPKC-CAAX show nuclear accumulation. Lamin and tubulin were used as nuclear and cytoplasmic controls, respectively. (E) Expression analysis using whole cell lysates from HeLa cells overexpressing various aPKC constructs as shown. GFP, control for transfection and loading. Scale bars: 50 μm in A; 250 μm in B. |
