Fig. 5: CHIP influences the interactions between β-catenin and LEF1 or GSK3β, as well as GSK3β and Arc.

A CHIP enhanced the interaction between β-catenin and LEF1. Immunoprecipitation and immunoblot analysis of HEK293T cells which were transfected with HA-LEF1, Flag-β-catenin, and Myc-CHIP, and stimulated with Wnt3a (40 ng/ml) for 1 h. B–D HEK293T cells were transfected with indicated plasmids or CHIP-specific siRNA as figures shown. Whole cell extracts were immunoprecipitated with anti-Flag beads and blotted with anti-HA antibody. CHIP heightened the endogenous interaction between β-catenin and LEF1 (B). Knockdown of CHIP reduced the interaction between β-catenin and LEF1 (C). The inactive mutant of CHIP could not enhance the interaction between β-catenin and LEF1 (D). E–F Immunoprecipitation and immunoblot analysis were performed to detect the endogenous interaction between β-catenin and LEF1. The indicated plasmids or CHIP-specific siRNA were transfected as figures shown in SHSY5Y cells. Overexpression of CHIP enhanced (E), while knockdown of CHIP reduced (F) the endogenous interaction between β-catenin and LEF1. G–H HEK293T cells were transfected with various combinations of plasmids including HA-Arc, Flag-GSK3β (G), HA-GSK3β, Flag-β-catenin (H), and Myc-CHIP, or Myc-CHIP T246M, and stimulated with Wnt3a (40 ng/ml) for 1 h. Immunoprecipitation and immunoblot analysis of CHIP regulated the interaction between GSK3β and Arc or β-catenin. All results were representative of three independent experiments.