Figure 3

Interaction between PTEN and AIB1. (A) MCF-7 cells were treated with 10 μM MG132 for 8 h, and then subjected to immunoprecipitation with anti-PTEN antibody followed by western blot analysis with anti-AIB1 antibody or vice versa. Immunoprecipitation carried out with anti-IgG antibody was used as control. (B) MCF-7 cells were treated with 10 μM MG132 for 8 h and then harvested for the preparation of nuclear and cytosolic extracts. These extracts were subjected to immunoprecipitation with anti-IgG or -PTEN antibody followed by western blot analysis with anti-AIB1 antibody. (C) COS-7 cells transfected with Flag-tagged AIB1 and Gfp-tagged wt or mu PTEN were treated with 10 μM MG132 for 8 h. The cells were then harvested and subjected to immunoprecipitation with anti-Flag antibody followed by western blot analysis with anti-Gfp antibody. (D) COS-7 cells were transfected with Gal4-DBD-tagged AIB1 and Flag-tagged PTEN full-length (FL), PTEN Δ1, PTEN Δ2 or PTEN Δ3 and then treated with 10 μM MG132 for 8 h. The cells were harvested and subjected to immunoprecipitation with anti-IgG or -DBD antibody followed by western blotting with anti-Flag antibody. A Clean-Blot IP Detection Reagent (HRP) from Thermo Scientific which eliminates the detection-interference of IP antibodies was used as secondary antibody. (E) COS-7 cells were transfected with Gal4-DBD-tagged AIB1 alone or together with Flag-tagged PTEN FL, PTEN Δ1, PTEN Δ2 or PTEN Δ3 and then subjected to western blot analysis with the indicated antibodies.