Figure 5: ABRO1 regulates p53 stability by inhibiting p53 ubiquitination.

(a) HCT 116 p53^+/+ cells were co-transfected with the indicated constructs. Twenty-four hours later, the cells were treated with 20 μM MG-132 for 4 h. Proteins were extracted, immunoprecipitated with p53 (FL-393) antibody and immunoblotted with anti-Ub antibody (left) or p53 antibody (DO-1) (right). (b) HCT 116 p53^+/+ cells were transfected with various ABRO1 siRNAs or with siNC. After 48 h, the cells were treated with 20 μM MG-132 for 4 h before harvesting and lysis for immunoprecipitation with p53 (FL-393) antibody and immunoblotting with anti-Ub antibody. (c) HCT 116 p53^+/+ cells were seeded onto small slide glass coverslips in 24-well culture plates and transfected with ABRO1 siRNAs or siNC. Forty-eight hours later, the cells were treated with 20 μM MG-132 for 4 h and the subcellular localization of p53 was detected by immunochemistry with p53 antibody. Scale bar, 10 μm. (d) HCT 116 p53^+/+ cells were treated as in c. The nuclear protein fractionation assay was performed, and the samples were immunoblotted with the indicated antibodies. (e) MEF mdm2^−/−/p53^−/− cells were co-transfected with Flag-ABRO1 and HA-p53 plasmids. Proteins were extracted and subjected to western blotting.