Determination of acetylated p53 levels

H1299 cells were transfected with p53 or p53/PCAF or p53/PCAF/HOPS. Twenty‐four hours after transfection, cells were treated with 10 μM trichostatin A (TSA, Sigma‐Aldrich) and 5 mM nicotinamide (NAM, Sigma‐Aldrich) for 8 h to prevent deacetylation. Then, the cells were lysed with RIPA buffer containing PIC, 1 mM PMSF, 10 μM TSA and 5 mM NAM with mild sonication. The protein lysates were precleared with G‐protein agarose beads for 1 h, and the total p53 immunoprecipitation was performed using anti‐p53 antibody (DO‐1) ON at 4°C. Next day, the immunoprecipitation was incubated with G‐protein agarose beads for 2 h at 4°C. The beads were eluted by Laemmli buffer after being washed with 1 ml of RIPA buffer three times. The samples were further tested by Western blot analysis.