Fig. 2: Altered expression of YOD1 affects p53 protein levels.

A U2OS and HCT116 cells were transfected with Flag-vector or Flag-YOD1 for 36 h, while AML cell lines MV-4-11 and MOLM13 cells were infected with control or YOD1 lentivirus for 96 h. Cell lysates were subjected to immunoblotting with specified antibodies. B U2OS and HCT116 cells were transfected with control or YOD1 siRNA for 36 h, and MV-4-11 and MOLM13 cells were infected with control or YOD1 shRNA lentivirus for 96 h. This was followed by immunoblotting assays. C, D U2OS cells were transfected with Flag-vector or Flag-YOD1 for 36 h and then treated with 50 μg/ml cycloheximide for the indicated periods of time before extraction. Relative protein levels of p53 were determined by densitometric analyses with β-actin as a reference. E, F Catalytic inactivation of YOD1 by the C160S point mutation diminishes its effects on p53 expression. U2OS cells were transfected with the catalytically inactive C160S mutant form of YOD1 and treated as above. Representative results are shown from triplicate experiments with similar results.