Fig 5.

Differential PB1-F2 expression is replicated in a plasmid model system. (A) Diagrams of plasmids created to examine PB1-F2 expression in transfected cells. A C-terminal 3×FLAG tag was inserted in frame with the PB1-F2 ORF in the pHW2000/PB1 or pDP2002/PB1 plasmid from PR8 (pPPP/3×FLAG), OH07 (pOOO/3×FLAG), MOM (pMOM/3×FLAG), and an Mx09 PB1 gene mutated to restore the PB1-F2 ORF (pMxKI/3×FLAG). (B) The indicated plasmids were transfected into MDCK cells, and at 24 h posttransfection, the cells were immunostained with antibodies against PB1 and 3×FLAG, followed by Alexa Fluor-conjugated secondary antibodies. The percentage of PB1-expressing cells also expressing PB1-F2/3×FLAG was determined and plotted on a graph. Statistically significant groups (P < 0.01) compared to PPP/3×FLAG are indicated with asterisks. The error bars indicate standard deviations. (C) The indicated plasmids were transfected into 293T cells, and at 48 h posttransfection, the cells were lysed and immunoprecipitated with antibodies against 3×FLAG or PB1. The immunoprecipitated proteins were separated on SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with 3×FLAG or PB1 antibodies. Lysates from the transfections were immunoblotted with antibodies against β-actin as a control for protein input levels in immunoprecipitation.