Fig 4.

PNR modulates p53 posttranslationally in HeLa cells. (A) PNR does not stimulate p53 transcription. Two days after transfection, p53 mRNA levels were measured by qRT-PCR and normalized to that of the sample with 0 μg of PNR. (B) PNR stabilizes p53 protein. Levels of endogenous p53 remaining after treatment with cycloheximide (2 μg/ml) for the indicated times were measured by immunoblotting, and the level in the 0-min sample was normalized to 1. Upper panel: GFP control transfection (1.2 μg). Middle panel: PNR transfection (1.2 μg). Lower panel: plot of remaining p53 protein levels versus treatment time. (C) PNR stimulates the specific activity of p53 as a transcriptional factor. Increasing amounts of the p53 expression plasmid were cotransfected with a constant amount of p53RE-FLuc to provide standard curves of p53 protein levels versus reporter activity. For both p53 protein measurement by immunoblotting (upper panel) and p53 transcriptional activity measurement by reporter assay (lower panel), the results determined under the conditions represented by sample 1 were normalized to 1. The middle panel shows the relative intensities of p53 immunoblotting signal (y axis) versus doses of p53 plasmid transfected (x axis). Sample numbers correspond to the conditions shown in the upper panel (sample 1, empty vector control; sample 2, PNR; samples 3 to 7, p53 standard curve). *, P < 0.05 (one-tailed test).