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. 2014 Nov 24;34(33):4300–4310. doi: 10.1038/onc.2014.359

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Copyright © 2015 Macmillan Publishers Limited

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MDM2 competes for binding of mutant p53 with p63, but promotes the binding of mutant p53 with p73. (a) Immunoprecipitation of p53 with DO-1 in HCT116 p53−/− cells transfected with 2μg TAp63α in combination with 2 μg p53wt, p53R175H, p53R273H, p53I254R and MDM2 C464A. P53, MDM2 and p63 expression was determined in the inputs and p63 and MDM2 expression was determined in precipitated material by western blot using DO-1, Ab1 (MDM2) and BC4A4 (p63) antibodies. (b) Immunoprecipitation of p53 with DO-1 in BXPC3 cells (three 10 cm plates) that were transfected with 3 μg MDM2 C464A or pcDNA. MDM2, p63 and p53 expression was determined in inputs and immunoprecipitations by western blot using p53 DO-1, MDM2 SMP14 and p63 4A4 antibodies. (c) Immunoprecipitation of p53 using DO-1 in HCT116 −/− cells transfected with 3 μg HA-TAp63α and p53R175H each in combination with 0, 0.5, 1.0, 2.0, 4.0 or 6.0 μg MDM2 C464A or compensatory amounts of pcDNA. MDM2, p63 and p53 expression levels were determined in the input and precipitated material using western blot and SMP14, HA and DO-1 antibodies, respectively. (d and e) Immunoprecipitation of p53 with DO-1 (d) or 1801 (e) in HCT116 p53−/− cells transfected with 2 μg HA-TAp73α (d) or HA-ΔNp73α (e) in combination with 2 μg p53wt, p53R175H, p53R273H, p53I254R, p53ΔI (only in e) and MDM2 C464A. P53, MDM2 and p73 (HA) expression was determined in the inputs and in precipitated material by western blot using DO-1 (d), 1801 (e), Ab1 (MDM2) and HA antibodies. (f) Immunoprecipitation of p53 with DO-1 in U251 cells that were transfected with 3 μg MDM2 C464A or pcDNA. MDM2, p73 and p53 expression was determined in inputs and immunoprecipitations by western blot using p53 DO-1, MDM2 SMP14 and a p73 antibody.