1. DNA damage signaling regulates mutant p53 ubiquitination.
DNA damage signaling regulates mutant p53 ubiquitination. A, WI38 and H1299 cell lysates were immunoblotted for phospho ATM. Actin was immunoblotted as a loading control. B, H1048 cells were treated with 2 mmol/L caffeine for 0 to 4 hours. Extract was harvested at 1 hour intervals and subjected to Western blotting (DO-1) with higher molecular weight portion of the gel exposed longer than the portion including native p53 to better reveal polyubiquitin conjugates. Actin was immunoblotted as a loading control. C, H1299 cells were transfected with empty vector (mock) or vectors encoding p53-273H, MDM2, and His-tagged ubiquitin. Forty to forty-eight hours posttransfection cells were treated with or without 2 mmol/L caffeine for 1 hour followed by addition of 2 μmol/L doxorubicin for 2 hours. Ubiquitinated proteins from cell lysates were pulled down using Ni-NTA beads and immunoblotted using anti-p53 antibody (DO-1). A fraction of the eluent was immunoblotted for his-ubiquitin with anti-ubiquitin antibody.
Rebecca A. Frum et al. Mol Cancer Res 2016;14:
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