Fig. 7. GPC4 modulates FGF signalling during neurulation. (A) Immunoblot analysis of GPC4/FGF2 complexes, as detected by anti-Myc antibodies. The `Input' lane contains NIH3T3 cells transfected with the Myc epitope-tagged mouse Gpc4 cDNA. The HS-GAG modified mouse GPC4 proteins have an apparent M<sub>r</sub> of around 200 kDa (arrow), whereas the unmodified proteins run at 60 kDa (asterisk). In the `GST-FGF-2' lane only the modified 200 kDa GPC4 protein (arrow) binds to FGF2. Mouse GPC4 does not bind to GST (control; `GST' lane). (B) Immunoblot analysis of phosphorylated ERK (pERK) and SMAD1 (pSMAD1) proteins in Xenopus embryos (stage 15). Levels of phosphorylated proteins were determined in embryos that were: cultured in the presence of the FGF inhibitor SU5402 (0.1 mg/ml; lane `SU5402'); cultured with DMSO as a control (lane `DMSO'); injected with CoMo (lane `CoMo'); injected with Gpc4Mo (lane `Gpc4Mo'); or co-injected with Gpc4Mo and mouse Gpc4 mRNA (lane `Gpc4Mo + mGpc4'). TUB, -Tubulin levels in the extracts were determined to normalize samples. (C-H) Molecular analysis of embryos cultured with DMSO (panels C,E,G) and with SU5402 (0.1 mg/ml; panels D,F,H). Arrows in panels C-F indicate Emx2 expression. (C) Emx2 expression in control embryos cultured with DMSO (stage 17). (D) Downregulation of Emx2 in embryos cultured with SU5402 (stage 17). (E) Emx2 and Krox20 (bracket) expression in control embryos cultured with DMSO (stage 22). (F) Downregulation of Emx2, but not Krox20 (bracket), in embryos cultured with SU5402 (stage 22). (G) Nkx2.1 expression in embryos cultured with DMSO (stage 22). (H) Nkx2.1 expression in embryos cultured with SU5402 (stage 22). (I) Lack of cell death in the forebrain region of an embryo cultured with DMSO (stage 20). (J) Apoptotic cells detected in the forebrain region of an embryo cultured with SU5402 (stage 20).

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