Fig. 5

Biochemical analysis of the Cav-1 and β-catenin interaction. The following assays were performed in ZF4 cells. Immunoprecipitation assays (IP) and Western blots (WB) were performed to detect interaction between Flag-Cav-1 (or its Flag-mutants) and HA-β-cat (or HA-β-cat-M). Data for luciferase activity assays represent mean ± S.D. from three independent experiments. **indicates p < 0.01 vs. cells transfected with Top-flash plus β-cat. TCL indicates total cell lysates. (A) Critical amino acid residues in Cav-1 scaffolding domain and in putative Cav-1 binding motif of β-catenin were highlighted in red. (B) Reciprocal interaction between Flag-Cav-1 and HA-β-cat. (C) Luciferase activity of Top-flash reporter was inhibited in a dose-dependent manner by co-transfection of 50 ng β-cat plasmid DNA and 100 ng Top-flash reporter DNA with Cav-1α plasmid DNA at 25 ng (+), 50 ng (++) and 75 ng (+++). WB analysis showed the proper expression of β-cat and the Cav-1 expression in a dose-dependent manner. (D) Amino acid residues deleted or substituted in Cav-1 scaffolding domain were underlined. (E) Mutations in Cav-1 affect reciprocal interaction between Flag-Cav-1 and HA-β-cat. (F) Activation of Top-flash reporter by transfection of 50 ng β-cat plasmid DNA was inhibited by co-transfection of 75 ng plasmid DNA for Cav-1α, Cav-1αΔDGVW or Cav-1α/F, but not by co-transfection of 75 ng plasmid DNA for Cav-1α/F + V or Cav-1α/F + T. WB analysis showed the proper expression of β-cat and Cav-1. (G) Amino acid residues substituted in putative Cav-1 binding motif of β-catenin were underlined. (H) Substitution of three amino acid residues in putative Cav-1 binding motif of β-catenin disrupted the interaction between HA-β-cat and Flag-Cav-1. (I) Activation of Top-flash reporter by transfection of 50 ng β-cat-M plasmid DNA was not inhibited by co-transfection of 75 ng Cav-1α plasmid DNA. WB analysis showed the proper expression of β-cat and Cav-1.