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Fig. 4. Stimulation of the signaling activity of LRP6 by oligomerization. (A) Schematic representation of wild-type and mutant LRP6. Signal peptide (SP), EGF repeats (E1-E4), LDLR repeats (L1-3) and transmembrane domain (TM) are indicated. In LRP6{Delta}EGF, all EGF repeats were deleted. In LRP6{Delta}N, the whole extracellular domain was deleted. Two copies of FKBPv were tethered to the C terminus of LRP6{Delta}EGF to form LRP6{Delta}EGF-FKBP. The extracellular domain of LRP6 was replaced by the extracellular domain of TrkC or human FZ5 to form TrkN-LRP6C and HFz5N-LRP6C. (B) AP20187 increases the stimulatory effect of LRP6{Delta}EGF-FKBP on LEF-dependent transcription in 293 cells. 293 cells were transfected with the indicated plasmids and CMV-LEF1, CMV-Renilla luciferase and a LEF-luciferase reporter. Cells were either treated with vehicles or 50 nM AP20187 for 36 hours. The levels of luciferase activities were normalized for Renilla luciferase activities. (C) AP20187 increases LRP6{Delta}EGF-FKBP-induced ß-catenin stabilization in 293 cells. 293 cells were transfected with the indicated plasmids and treated with vehicles or 50 nM AP20187 for 36 hours. Cells were subjected to subcellular fractionation, and cytosolic ß-catenin was determined by immunoblotting. (D) AP20187 increases the binding of Axin to LRP6{Delta}EGF-FKBP during ß-catenin stabilization in 293 cells. 293 cells were transfected with the indicated plasmids and treated with vehicles or 50 nM AP20187 for 36 hours. Proteins were immunoprecipitated from cell lysates with anti-HA antibodies, and immunoprecipitates were fractionated by SDS-PAGE, transferred to a nitrocellulose membrane and immunoblotted with anti-Myc antibodies (upper panel). Expression of Axin and LRP6{Delta}EGF or LRP6{Delta}EGF-FKBP in total cell lysates was examined by immunoblotting with anti-Myc and anti-HA antibodies (lower panels). (E) NT3 increases the signaling activity of TrkN-LRP6C in S2 cells. S2 cells were transfected with the indicated plasmids and treated with 200 ng/ml NT-3 (Upstate) for 48 hours. Cells were lysed and the luciferase activities were normalized for Renilla luciferase activity. (F) The signaling activity of LRP6, with or without inducible oligomerization, is Dsh independent. S2 cells were treated with dsRNAs, transfected with the indicated effector plasmids and treated with 200 ng/ml NT3 for 48 hours. Cells were lysed and luciferase activities were measured.