FIG. 3.

Expression of HPV-16 E7 and NPT mRNA and protein. HeLa cells growing on 6-cm plates (3 × 10⁵ cells/plate) were transfected with the GFP-expressing plasmid pQBI-25-fPA (−) alone or cotransfected with this plasmid and a plasmid carrying either synthetic (pIn2-eE7) or wild-type (pIn2-E7) E7. The latter plasmids are transcribed into bicistronic mRNAs harboring sequences for expression of the E7 and NPT proteins. After 24 h, cells were harvested and lysates were processed for total or cytoplasmic RNA purification and for protein extraction. (A) Western blot analysis. Note the remarkably small amount of E7 protein expressed in cells transfected with pIn2-E7 compared with that in cells transfected with pIn2-eE7. In contrast, the difference in the amount of NPT expressed by the two plasmids is much lower (about 1.5 times higher by pIn2-eE7, as estimated densitometrically). Hybridization with GFP antibodies showed equal efficiency of transfection. (B) Northern blot analysis showing levels of eE7-IRES-NTP and E7-IRES-NTP bicistronic RNA in transiently transfected cells. Total RNA was loaded at 10 μg per lane. Detection was with [α-³²P]dCTP-labeled probes. A labeled NPT probe was used to detect the eE7-IRES-NPT and E7-IRES-NPT messages (arrowhead). The blot was stripped and rehybridized with a radioactive GFP probe to show nearly equal transfection efficiencies (middle). Staining of the gel with ethidium bromide after electrophoresis is shown in the lower panel. The positions of the 28S (4.7-kb) and 18S (1.9-kb) rRNAs are indicated.