Figure 4
Effect of PTEN on the interaction between Fbw7 α and AIB1. (A) COS-7 cells transfected with Flag-tagged Fbw7α and Gfp-tagged PTEN were subjected to immunoprecipitation with anti-Flag antibody followed by western blot with anti-Gfp antibody or vice versa. (B) COS-7 cells transfected with Flag-tagged Fbw7α and Gfp-tagged wt or mu PTEN were subjected to immunoprecipitation with anti-Flag antibody followed by western blot with anti-Gfp antibody. (C) COS-7 cells transfected with Gfp-tagged Fbw7α and Flag-tagged PTEN FL, PTEN Δ1, PTEN Δ2 or PTEN Δ3 were collected and then subjected to immunoprecipitation with anti-Gfp antibody, followed by western blot analysis 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. (D) COS-7 cells were transfected with Flag-tagged AIB1 and Gfp-tagged wt or mu PTEN and with or without siFbw7α. Cells were collected and subjected to western blot analysis with the indicated antibodies. (E) 293T cells were transfected with Flag-tagged Fbw7α with or without Gfp-tagged PTEN or siPTEN as indicated, and then treated with 10 μM MG132 for 8 h. Cells were collected and then subjected to immunoprecipitation with anti-AIB1 antibody followed by western blot with anti-Flag antibody. ★ indicates overexpressed Gfp-tagged PTEN. (F) 293T cells were transfected with Flag-tagged Fbw7α with or without wt or mu PTEN as indicated, and then treated with 10 μM MG132 for 8 h. The cells were harvested and subjected to immunoprecipitation with anti-AIB1 antibody followed by western blot with anti-Flag antibody. (G) 293T cells transfected with various combinations of different constructs as indicated were treated with 10 μM MG132 for 8 h. The cells were collected and subjected to immunoprecipitation with anti-AIB1 antibody followed by western blot analysis with anti-Myc antibody.