Figure 1

Deletion of the PS domain of BMI1 results in increased half life of BMI1. (A). Schematic representation of wild type BMI1 and its deletion mutants. The domain structure of the wild type BMI1, and the ∆HT and ∆RF mutants has been described previously [34, 35]. The ∆PS mutant of BMI1 encodes a protein, which lacks amino acids 236- 326. Its cDNA was generated by PCR and cloned in the pBabe- puro retroviral vector. (B). The half life of wild type and mutant BMI1 in MCF10A cells expressing the respective protein was determined by blocking synthesis of new proteins using 100 μg/ml CHX for different time points (0-60 min) and the rate of BMI1 degradation was determined by western blot analysis of remaining BMI1 protein after each time point and normalizing it to ß-actin using densitometry measurements as described in the Materials and Methods section. In case of the wild type, and the ΔRF and the ΔHT mutants, only the lower most band of BMI1, which showed a time-dependent decrease was quantified to determine the half life. The densitometric analysis was performed using Image J software (NIH, Bethesda, MD), and the graph was generated by plotting % residual protein vs time of CHX treatment.