p53 degradation assays

In vitro p53 degradation assays were performed in a 30 μL reaction in assay buffer (25 mM Tris HCl pH 7.5, 100 mM NaCl, 2 mM DTT, 2.5 mM ATP) containing 1 μl rabbit reticulocyte lysate (RRL), p53, E6 proteins or E6 buffer. Reactions were incubated for 2h at 25°C unless otherwise stated and were stopped by incubation at 75°C. For inhibition studies, E6 proteins were diluted to 0.1μM and equilibrated with assay buffer containing 0.35% (v/v) DMSO, or compounds at different concentrations for 1h, followed by the addition of p53 and RRL and incubation at 25°C for 2h. Proteins were separated on 10% SDS page, transferred onto PVDF membrane and blotted for p53 using Pab1801 antibody. Blots were stripped and re-probed with anti-HPV-16 E6 antibody (ArborVita Inc.). All p53 degradation experiments were repeated at least three independent times.

The p53 in vivo degradation assay was performed as described with the following modifications [40]. C33A cells were transfected with pRluc-p53 (215 ng), pCI-Fluc (135 ng), HPV-16 E6 expression vectors (0, 18.5, 54, 270, 540, 1080 ng) and carrier DNA (up to 1080 ng, pBabePuro) using Lipofectamine 2000 (Invitrogen) with OptiMem media. 24h later, cells were lysed using the Dual Glo Luciferase Assay System (Promega) and each 6-well sample was aliquoted into four replicates into a 96 well plate. Renilla luciferase levels were normalized to firefly luciferase. The averages of the three independent experiments were plotted using GraphPad Prism.