Figure 3.

BMI1 copurifies with the NHEJ proteins. A, 293T cells were transfected with the EFv-CMV-GFP (GFP) or EFv-BMI1-Myc-CMV-GFP (BMI1-Myc) plasmids. Transfected cells were exposed to an ATM kinase inhibitor (ATMi; 10 μm) or DMSO for 1 h, and irradiated (3 Gy) or left untreated. Twenty minutes after IR, whole-cell homogenates were immunoprecipitated with an anti-Myc antibody and subjected to Western blot analyses. The asterisk marks a notable reduction in total ATM IP upon cells exposure to ATMi. The cell lines used were GBM0611 and GBM0811. B–D, GBM cells were infected with the EFv-CMV-GFP (GFP)- or EFv-BMI1-Myc-CMV-GFP (BMI1-Myc)-carrying lentiviruses. Infected cells were sorted into CD133− and CD133+ fractions and irradiated (3 Gy, 20 min). Protein extracts were subjected to IP using an anti-Myc antibody. B, Immunoprecipitates were resolved by SDS-PAGE and revealed by silver staining. Indicated protein bands were excised from the gel, digested with trypsin, and analyzed by LC-MS. C, List of proteins immunoprecipitated with BMI1-Myc as identified by LC-MS. D, Immunoprecipitates were resolved in SDS-PAGE and analyzed by Western blot. Note the preferential copurification of BMI1 with NHEJ proteins in CD133+ cells.