FIG. 12.

Comparison of the capacity of wild-type ORF50 and the ORF50(KK/EE) mutant to activate PAN expression in HH-B2 cells. (A) Expression of ORF50 and ORF50(KK/EE) was analyzed by immunoblotting with an ORF50 polyclonal antibody at different times after transfection of HH-B2 cells with 10 μg of ORF50 or ORF50(KK/EE) expression plasmid. (B) Extracts of HH-B2 cells harvested 12 h after transfection were used in EMSA with PANp as a probe. Antibodies to ORF50(230-250) and Sp1 were used for the supershift assay. (C) Activation of PAN RNA by ORF50 and ORF50(KK/EE). Total RNA was prepared from transfected HH-B2 cells at different time points, and expression of PAN RNA was analyzed by Northern blot hybridization. Hybridization with H1 RNA of RNase P served as a loading control.