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. 2012 Feb 8;287(13):10013–10020. doi: 10.1074/jbc.M111.335141

FIGURE 4.

FIGURE 4.

MPA induces NS degradation independently of MDM2. A, NS polyubiquitination occurs independently of MDM2. p53−/−/MDM2−/− MEF cells were transfected with or without a His-ubiquitin (Ub) expression plasmid. Twenty-fours hours after transfection, cells were treated with 10 μm MG132 for 6 h. Cell lysates were subjected to His-nickel bead pulldown assay, followed by SDS-PAGE and Western blotting to detect polyubiquitinated NS species and input. B, NS degradation can be rescued by MG132 independently of MDM2. p53−/− or p53−/−/MDM2−/− MEF cells were treated with 10 μm MG132 for 8 h and harvested, followed by SDS-PAGE and Western blotting. C, MPA induces NS degradation independently of MDM2. p53−/− or p53−/−/MDM2−/− MEF cells were treated with 40 μm MPA and harvested at the indicated time points. Cell lysates were analyzed by SDS-PAGE and Western blotting with the indicated antibodies. D, MDM2-independent decrease in NS levels upon treatment with various reagents. p53+/+ and p53−/− HCT116 cells were treated with Me2SO as a control (Cont), 5 nm actinomycin D (ActD), 40 μm MPA, 1 mm doxorubicin (Dox), and 10 μm etoposide (Etop) for 12 h and harvested for Western blot analyses using the indicated antibodies.