Figure 2

Characterization of the interaction between PTEN and BMI1 proteins. (A) PTEN binds to BMI1 independently of its phosphatase activity. 293T cells were transiently transfected with BMI1, PTEN, PTEN(C124S) (C124S), PTEN(G129E) (G129E), a C-terminal PTEN fragment (residues 186-403) (C-PTEN) for 48 hours. Cell lysates were prepared and immunoprecipitated with anti-PTEN and anti-FLAG (M2) (for ectopic BMI1) antibodies. The precipitates and lysates were analyzed by western blot using the indicated antibodies. The # and * symbols indicate endogenous PTEN and a possible oligomer of C-PTEN, respectively. (B) Mapping the BMI1 binding motif of the PTEN protein. A set of PTEN truncation mutants were constructed. Their interaction with BMI1 was examined. C2: C2 domain. The + and - symbols indicate binding or not-binding of individual PTEN proteins to BMI1. (C) C2 binds to BMI1. FLAG-tagged BMI1 and HA-tagged C2 were transfected into 293T cells as indicated. BMI1 was immunoprecipitated with an anti-FLAG antibody (M2) or a control IgG (IgG), followed by western blot examination for BMI1 and C2. 20% of the cell lysates that were used for immunoprecipitations were also analyzed. The * symbols indicate background bands. (D) C2N binds to BMI1. C2N, C2C, and C-tail (left panel) and their GFP fusion counterparts (right panel) were co-transfected with either an empty vector (-) or FLAG-tagged BMI1 as indicated, followed by immunoprecipitation with M2 or control IgG (IgG) and then western blot (WB) with the indicated antibodies. The respective cell lysates were shown at the bottom panels.