FIG. 3.

Expression of PB1 mutants in primate cells. WT and mutant PB1 were expressed in CV-1 cells infected with vaccinia-T7 and transfected with expression plasmids. Cells were harvested at 18 hpi, and lysates were analyzed by Western blotting with a monoclonal antibody to the HA epitope tag (12CA5). WT and representative mutants of PB1 are shown (D2V, V3D, N4D, L7D, L8D, F9D, L10D, and A14D). A lysate from nontransfected cells was used as a negative control (−), while a lysate expressing an unrelated HA-tagged protein of 56 kDa served as positive control (+). Proteins were separated on an SDS–10% PAGE gel and transferred to a Hybond-C membrane (Amersham). The luminescent signal (ECL; Amersham) was recorded by exposure to X-ray film, which was subsequently developed, scanned, and digitized using Adobe Photoshop software. Small arrowheads indicate the two PB1 polypeptides that reacted with the anti-HA antibody.