Figure 5: Acetylation of lysines at the CTD of p53 suppresses the interaction with Fbxo22.

(a) p53^−/− HCT116 cells expressing Dox-inducible Flag-Fbxo22 together with wild-type EGFP-p53 (Wt) or a mutant p53 lacking the C-terminal 30 amino acids (Δ30) were incubated for 48 h in the presence or absence of doxycycline (1 μg ml⁻¹) and were then treated with MG132 (10 μg ml⁻¹) for 2 h. The lysates were immunoprecipitated using anti-Flag M2 affinity gel. The resultant immunoprecipitates and lysates (Input) were subjected immunoblotting using the indicated antibodies. (b) p53^−/− HCT116 cells expressing Dox-inducible Flag-Fbxo22 together with the wild-type or the indicated mutants of EGFP-p53 were incubated for 48 h in the presence or absence of doxycycline (1 μg ml⁻¹) and were then treated with MG132 (10 μg ml⁻¹) for 2 h. The lysates were immunoprecipitated using anti-Flag M2 affinity gel. The resultant immunoprecipitates and lysates (Input) were subjected to immunoblotting using the indicated antibodies. (c) RPE cells expressing the Dox-inducible shControl or shFbxo22 were incubated for 48 h in the presence of doxycycline (1 μg ml⁻¹) and were then treated with MG132 (10 μg ml⁻¹) for 2 h. The lysates were immunoprecipitated using a control IgG, methyl (K370me2)- or acetyl (K370Ac)-specific anti-p53, or anti-p53 antibodies. The resultant immunoprecipitates and lysates (Input) were subjected to immunoblotting using the indicated antibodies.