Samples are crude lysates of HCT116 cells: Left lanes are control. Right lanes are cells treated with siRNA to knockdown the expression of a Tip60 interacting protein UHRF1, which results in an increase in acetylation of p53 at Lys120. Total p53 was immunoprecipitated with anti-p53 monoclonal antibody from the crude extracts and analyzed by western blotting with another anti-p53 antibody (upper panel) and ab78316, at 1µg/ml (middle panel). The lower panel shows total p53.

Overlay histogram showing HeLa cells stained with ab78316 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab78316, 1/100 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

Immunocytochemistry/Immunofluorescence analysis of HeLa cells treated with 100 nM Doxorubicin for 24 hour labelling p53 (acetyl K120) with ab78316 at a dilution of 1/1000. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.25% Triton X-100 in PBS for 10 minutes. DAPI was used to stain the nucleus.

Crude whole cell extracts were prepared from H129 cells (p53 negative cell line) expressing only Myc-p53 (first lane), and both Myc-p53 and His-Tip60 (Second lane). In the upper panel, the cell extracts were immuno-blotted with anti-Myc, anti-His-tag or anti-α-tublin antibodies. In the lower panel, the extracts were immuno-precipitated with ab78316 and the precipitates were immuno-blotted with anti-Myc antibody. Acetylation of p53 at K120 is dependent on Tip60 and promoted by over-expression of His-Tip60.