Degradation of p53 wild type and indicated mutants via E6.

<p>(A) Radiolabeled p53 (wild type and indicated mutants) and HPV16 E6 proteins produced in rabbit reticulocyte lysates were incubated together at 28°C. Aliquots were removed at the indicated times before separation by 12% SDS-PAGE and exposition to photographic film. (B) Same experiment as in A, but levels of radiolabeled p53, after exposition to photographic film, were quantified by densitometry (BIO-RAD, Quantity One Software). Mean values (AU) ± standard deviation for three independent experiments are shown. (C) H1299 cells were co-transfected by vectors for transient expression of HPV16 E6 and p53 proteins. Where indicated, 4 h prior to harvesting, the medium was supplemented with the 26S proteasome inhibitor ALLN at a final concentration of 100 µM. 24 h after transfection, extracted proteins were separated by 12% SDS-PAGE and analysed by Western-blotting using monoclonal anti-p53 antibody, polyclonal anti-actin antibody and monoclonal anti-16-E6 antibody.