Fig. 4.

Phospho-mutant BMI1 is intrinsically unstable. a Determining the half-life of Wild-type (WT) and S110A mutant BMI1 (Mut-BMI1). The half-life of wild-type and mutant BMI1 in CP20 cells expressing the respective protein was determined by blocking synthesis of new proteins using 100 μg/ml CHX for different time points (0–45 min) and the rate of BMI1 degradation was determined by western blot analysis of FLAG(BMI1) with HSP60 serving as a loading control. Residual BMI1 protein after each time point was normalized to HSP60 using densitometry measurements. The time-dependent decrease of BMI1 was quantified to determine the half-life and is graphically represented in the right panel. The densitometry analysis was performed using Image J software (NIH, Bethesda, MD), and the graph was generated by plotting % residual protein vs time of CHX treatment. b Effect of proteosomal inhibition on Wild-type (WT) and S110A mutant BMI1 (Mut-BMI1). 30 h post transfection, CP20 cells were treated with 10 μM MG132, for different time points (0–120 min) to determine the accumulation of WT or mutant BMI1. Left panel represents the immunoblots for FLAG and HSP60 and quantification of the accumulated proteins by densitometry analysis of signal present in respective lanes (normalized to the individual HSP60) is graphically represented in the right panel. c Effect of proteosomal inhibition on BMI1 protein accumulation in CK2α silenced cells. CP20 cells were transfected with scrambled siRNA (siCTL) or siRNA against CK2α (siCK2α) and 48 h post transfection, cells were treated with 10 μM MG132, for different time points (0–120 min) to determine the effect of silencing CK2α on MG132 induced accumulation of endogenous BMI1. siCTL or siCK2α transfected cells were harvested after each time point and analyzed for accumulation of the endogenous BMI1 protein by western blot analysis and using HSP60 as a loading control (bottom left panel). Efficient silencing by CK2α siRNA was also determined (top left panel). The accumulated proteins were quantified by densitometry analysis of signal present in respective lanes and by normalizing it to the individual HSP60 and graphically represented in the right panel