A, Structure of HLI98 family and HLI373. HLI98C: R2 = Cl, HLI98D: R1 = Cl, HLI98E: R1 = CH3. B, RPE cells were treated with 1 μg/ml Adriamycin (Adr), 50 μM MG132 (MG), or HLI373 dissolved in PBS or DMSO and added to cells at the indicated final concentrations for 8 h. Levels of p53, Hdm2 and β-actin, used as a loading control, were assessed by immunoblotting with antibodies directed against the indicated proteins. C, RPE cells were incubated with 1 μg/ml Adriamycin, 50 μM ALLN, or the indicated concentrations of HLI373 for 8 h followed by assessment as in B. D, Immobilized bacterially-expressed GST-Hdm2 was treated as indicated prior to carrying out an in vitro auto-ubiquitylation reaction using 32P-labeled ubiquitin. Lack of E1 in the reaction mixture served as a negative control.