Figure 4: p53 interacts with Fbxo22 in a phosphorylation-independent manner.

(a) 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 cell lysates were immunoprecipitated using anti-Flag M2 affinity gel. The resultant immunoprecipitates and lysates (Input) were subjected to immunoblotting using anti-Flag or anti-p53 antibodies. (b) RPE cells were treated with MG132 (10 μg ml⁻¹) for 2 h and the lysates were subjected to immunoprecipitation using the anti-p53 antibody or a control IgG. The resultant immunoprecipitates and lysates (Input) were subjected to immunoblotting using the indicated antibodies. (c) Phosphorylation of p53 did not affect its interaction with Fbxo22. 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 or without MG132 (10 μg ml⁻¹) for 2 h and/or with IR (6 Gy). The lysates were immunoprecipitated using anti-Flag M2 affinity gel. After immunoprecipitation, some samples were treated with protein phosphatase (PP) for 1 h. The resultant immunoprecipitates and lysates (Input) were subjected to immunoblotting using the indicated antibodies. The asterisk indicates an input sample before PP treatment. (d) Interaction between Fbxo22 and p53 was suppressed by treatment with TSA. 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 or without TSA (1 mM) for 12 h and/or MG132 (10 μg ml⁻¹) for 2 h. The cell lysates were immunoprecipitated using anti-Flag M2 affinity gel. The resultant immunoprecipitates as well as lysates (Input) were subjected to immunoblotting using the indicated antibodies.