Figure 5. DDB2 degradation is dependent on PCNA binding. (A) HeLa cells expressing DDB2^wt or DDB2^mut proteins were irradiated (30 J/m²) and collected 0.5, 4, 7, and 14 h later. Parallel samples of cells transfected with DDB2^wt plasmid were also treated with MG132 proteasomal inhibitor and collected at the same times. The samples were fractionated, and the soluble form was analyzed by western blot for DDB2 content. In the same samples, actin levels, reported as loading control, are also shown. (B) DDB2 protein levels in similar experiments, as those shown in (A), were quantified by densitometric analysis and normalized to actin values, as standard for protein loading. The mean values (± s.d.), obtained from 3 independent experiments are reported. Statistical analysis was performed by t test. *P < 0.05; **P < 0.01. (C) After transfection with PCNA siRNA, HeLa cells were UV irradiated, incubated for 4 h, and analyzed for DDB2 degradation.