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. 2004 Dec;78(24):13769–13778. doi: 10.1128/JVI.78.24.13769-13778.2004

FIG. 5.

FIG. 5.

The p21WAF1/CIP1 gene is down-regulated in E6-expressing cells. (A) Analysis of p21WAF1/CIP1 mRNA levels by Northern blotting. Total RNA was prepared as described in Materials and Methods. Five- and 15-μg amounts of total RNA from each cell population were loaded onto a formaldehyde agarose gel, blotted onto a nylon membrane, and incubated with a 32P-labeled p21WAPI/CIPI DNA gene fragment. After being washed, the membrane was exposed to Kodak film for 12 h at −80°C. (B) p21WAF1/CIP1 levels are lower in E6-expressing cells than in the control cells. Total protein extracts (50 μg) from cell populations infected with the different recombinant retroviruses indicated were applied to an SDS-10% polyacrylamide gel, transferred onto a PVDF membrane, and incubated with an anti-p21WAF1/CIP1 or -β-tubulin antibody. (C and D) An HPV16 E6 mutant unable to target p53 efficiently decreases p21WAF1/CIP1 levels. Total protein extracts (50 μg) from the cell populations infected with the different recombinant retroviruses indicated were applied to an SDS-10% polyacrylamide gel, and Western blot analysis was performed as described for panel B with an anti-p21WAF1/CIP1, -p53, or -β-tubulin antibody.