Figure 6
Genotoxic stress-mediated perturbation of SMAR1–Cdc20 interaction leads to SMAR1 stabilization. (a) SMAR1 isstabilized upon genotoxic stresses. MCF7 cells were treated with either 5 μM CPT (Camptothecin) or IR (10 Gy), UV (10 J/m2) for 4 h. Cells were harvested, lysed and whole cell lysates were immunoblotted with indicated antibodies. (b) Cdc20 fails to degrade SMAR1 upon IR treatment. MCF7 cells were transfected either with vector or Cdc20 for 36 h. Transfected cells were then treated with and without IR for 4 h. Cells were collected, lysed and analyzed by western blotting using indicated antibodies. (c) Cdc20 fails to interact with SMAR1 upon DNA damage. Irradiated and un-irradiated MCF7 cell lysates were immunoprecipitated either with SMAR1 or Cdc20 antibody. Immunoprecipitates were analyzed by immunoblotting with indicated antibodies. (d) K48-linked polyubiquitylation level of SMAR1 was suppressed upon DNA damage. MCF7 cells were either untreated or treated with 5 μM CPT or 10 Gy IR for 4 h. Cell lysates were prepared and used for immunoprecipitation with anti-SMAR1 antibody. Immunoprecipitates were analyzed by immunoblotting using K48-linked ubiquitin antibody. (e) ATM regulates the interaction of SMAR1 and Cdc20.MCF7 cells were either untreated or irradiated with 10 Gy IR in the absence and presence of ATM inhibitor (KU55933). Cell lysates were prepared and immunoprecipitated with anti-SMAR1 antibody and analyzed by immunoblotting with indicated antibodies
