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Fig. 5. CTGF interacts with the WNT receptor complex. In all experiments, proteins were provided in the form of conditioned medium, prepared as described in Materials and methods. Media were mixed as indicated, precipitated by Protein A Sepharose beads, and visualised by immunoblotting with the indicated antibodies. (A) CTGF interacts with LRP6 and Frizzled8 extracellular domains (LRP6N and FzCRD) under low stringency conditions (150 mM NaCl). Binding to LRP6, but not to Frizzled8, resists more stringent washing (500 mM NaCl) (top panel). Input levels of CTGF (middle panel) and the IgG-tagged proteins (bottom panel) are shown. (B) The CT domain is required for the interaction of CTGF with LRP6. CTGF but not CTGF{Delta}CT is precipitated by LRP6N (top panel). Levels of CTGF and CTGF{Delta}CT (middle panel) and of control IgG, Fz8CRD and LRP6N (bottom panel) prior to immunoprecipitation are shown. (C) CTGF binds to both EGF repeats 1, 2 and 3, 4 of LRP6. LRP6 extracellular domain (LRP6N) and two deletion constructs, LRP6N{Delta}1,2 and LRP6N{Delta}3,4, all interacted with CTGF whereas control IgG did not (top panel). Note that whereas CTGF protein levels are comparable in all lanes prior to immunoprecipitation (middle panel), LRP6N{Delta}3,4 protein levels are lower than both LRP6N and LRP6N{Delta}1,2 (bottom panel), indicating that the interaction of CTGF with this deletion is stronger than appears in the top panel. (D) DKK1 interacts with EGF repeats 3 and 4 of LRP6. DKK1 binds to LRP6N and LRP6N{Delta}1,2, but not to LRP6N{Delta}3,4 (top panel). Dkk-(middle panel) and IgG-tagged protein levels (bottom panel) prior to immunoprecipitation are shown.