FIG 3.

Knockdown of E6AP and overexpression of p53 in p53-null H1299 cells lead to increased E6 phosphorylation. (A) H1299 cells were transfected with siE6AP and si-Luci (as control) using Lipofectamine RNAiMax. After 48 h, the same cells were transfected with plasmids expressing Flag-tagged p53, HA-tagged 18E6 (wild type), and HA-tagged 18E6 delPBM, together with a LacZ expression plasmid (transfection efficiency control), followed by further incubation of 24 h. Protein analysis was performed by Western blotting using specific antibodies against p53, p18E6, and HA (for probing 18E6). β-gal was used as a loading control for the overexpressed proteins, while α-tubulin was used as a loading control for E6AP siRNA knockdown. (B) The histogram represents the statistical significance of changes in pE6 in the absence of E6AP and presence of overexpressed p53 protein from at least three independent experiments. **, P value < 0.005; ns, nonsignificant. Statistically quantified using Student's t test; error bars indicate the standard deviation of the mean.