Figure 3.

BVES interacts with LRP6 and LRP5. (A) Reciprocal co-immunoprecipitation of Flag-tagged BVES (Flag-BVES) and GFP-tagged LRP6 (GFP-LRP6). HEK293T cells were transiently transfected with 4 μg of each plasmid using polyethylenimine. Filler plasmid was used to maintain equal DNA quantities. GFP-LRP6 was immunoprecipitated with GFP-binding protein magnetic beads following by immunoblotting with anti-Flag antibodies. Flag agarose resin was used to immunoprecipitate BVES followed by immunoblotting with anti-GFP antibodies. (B) Immunoprecipitation of Flag-BVES followed by immunoblotting for endogenous LRP5 and LRP6. 4 μg of vector or Flag-BVES was transfected into HEK293T cells using polyethylenimine. The LRP5 and LRP6 blots were developed using enhanced chemiluminescence and film, whereas the rest of the blots were immunoblotted using Odyssey infrared reagents as discussed in Materials and methods. (C) Schematic of the N-terminal myc-tag LRP6 (Myc-LRP6ICD) construct that spans amino acids 1364–1539 and contains the LRP6 transmembrane domain (TM) and proximal intracellular domain (ICD). Myc-LRP6ICD was transfected into HEK293T cells along with Flag-BVES as in (A) and immunoprecipitation experiments performed 48 h later. (D) Cells were transfected with 4 μg of Flag-BVES and then 48 h later treated with 50% L-cell-conditioned media or 50% Wnt3a-conditioned media for 2 h before flag immunoprecipitation.