Figure 6. MDMX C-terminus is essential for UbcH5c binding and p53 degradation.

(A) U2OS cells were transfected with empty vector DNA, MDMX or MDMX^ΔC7 plasmid DNA. IP was performed with an anti-MDMX antibody and blotted for the indicated proteins. (B) U2OS cells were transfected with vector, MDM2 or MDM2^XC7 plasmid DNA. IP was performed with an anti-MDM2 antibody and blotted for with the indicated proteins. (C) 2KO (Mdm2^−/−;p53^−/−) MEFs were co-transfected with p53 and MDM2 or MDM2^XC7 plasmid DNA. The levels of MDM2, p53 and actin were analyzed by western blot. (D) 2KO MEFs were co-transfected with vector, MDMX^ΔC7 or MDM2^XC7 plasmid DNA along p53 DNA. The cell lysates were isolated and immunoprecipitated with an anti-p53 antibody and analyzed by western blot using an anti-ubiquitin antibody. (E) 2KO MEFs were treated with siRNA against MDMX or non-specific sequences (NS) for 24 hours followed by transfection with indicated plasmid DNA for another 24 hours. Cell lysates were blotted for the indicated proteins. (F) 2KO MEFs were treated with siRNA against UbcH5c or non-specific sequences (NS) for 24 hours followed by transfection with indicated plasmid DNA for another 24 hours. Cell lysates were blotted for the indicated proteins.