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. 2003 Aug;23(16):5825–5835. doi: 10.1128/MCB.23.16.5825-5835.2003

FIG. 5.

FIG. 5.

Forced dimerization of the GyrB-LRP6 chimeric receptor inhibits LRP6 intracellular domain functions. (A) Schematic representation of GyrB-FGFR and GyrB-LRP6 constructs, in which the extracellular domains of FGFR2 and LRP6 were replaced by the amino-terminal 24K subdomain of the bacterial DNA gyrase B subunit (GyrB). TM, transmembrane domain; SP, signal peptide. (B) Coumermycin activates the GyrB-FGFR chimeric receptor. At 24 h following transfection of 293T cells with 0.5 μg of GyrB-FGFR, coumermycin was added at the concentrations indicated. Around 16 h later cell lysates were obtained and GyrB-FGFR was immunoprecipitated (IP) with anti-Flag antibody, following SDS-PAGE and immunoblotting with anti-p-Tyr. The same blot was stripped and reprobed with anti-Flag antibody. Normalized quantification of the immunoblots is shown at the bottom (Relative Ratio). DMSO, dimethyl sulfoxide. (C) Coumermycin inhibits constitutive β-catenin stabilization by GyrB-LRP6. Around 0.5 μg of GyrB-LRP6 or LRP6ΔE1-4 control plasmid was transfected into 293T cells. The conditions of coumermycin treatment were the same as those for panel B. Uncomplexed β-catenin analysis was performed as described in the legend to Fig. 1B. Quantified representation of the immunoblot is shown at the bottom of each blot (Relative intensity). (D) Coumermycin inhibits constitutive TCF signaling of GyrB-LRP6. At 6 h following transfection of 293T cells with 50 ng of GyrB-LRP6 or LRP6ΔE1-4 in 6-well dishes, coumermycin was added as indicated for 18 h. TCF-dependent transcription was measured as described in the legend to Fig. 1C. (E) Coumermycin inhibits the interaction between GyrB-LRP6 and Axin. At 48 h following transfection of 293T cells with 0.25 μg of Axin-myc, 1 μg of GSK3β, together with 0.5 μg of either Flag-LRP6ΔE1-4 or Flag-GyrB-FGFR, coumermycin was added at the concentration indicated for 30 min, and immunoprecipitation with anti-Flag antibody and immunoblot (IB) with anti-myc antibody were performed as described in the legend to Fig. 1D.