Figure 6: SCF^Fbxo22 forms a ternary complex with p53 and KDM4A that targets methylated p53 for degradation.

(a) Schematic representation of the domain structure of Fbxo22. (b) RPE cells expressing Dox-inducible wild-type Flag-Fbxo22 (Wt), and its mutants lacking FIST-N (ΔFIST-N) or FIST-C (ΔFIST-C) 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 Dox-inducible Flag-Fbxo22 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 subjected to sequential immunoprecipitation using anti-Flag M2 affinity gel and anti-KDM4A antibodies. The resultant immnoprecipitates and lysates were subjected to immunoblotting using the indicated antibodies. (d) RPE cells expressing the Dox-inducible shControl or shFbxo22 together with wild-type KDM4A (Wt) or its catalytically inactive mutant (H188A) 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 subjected to immunoblotting using the indicated antibodies. (e) RPE cells expressing the Dox-inducible shControl, shKDM4A or shKDM4A+shp53 were treated with doxycycline (1 μg ml⁻¹) and their relative numbers were determined at the indicated times. Data are presented as means±s.d. of at least three independent experiments. (f) RPE cells expressing the Dox-inducible shControl or shFbxo22, together with wild-type Flag-PHF20 (Wt) or its WD domain mutant (W97A/Y103A) 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 cell lysates were immunoprecipitated using anti-Flag M2 affinity gel. The resultant immunoprecipitates and lysates (Input) were subjected to immunoblotting using the indicated antibodies. (g) Schematic representation of CTD modifications that protect p53 from SCF^Fbxo22-targeted degradation.