Fig 7.

PB1 nucleotides 267 to 343 and 582 to 816 are necessary and sufficient for translational regulation of PB1-F2. (A to C) Diagrams and results for plasmids used to map sequences necessary and sufficient for PB1-F2 translational regulation. MDCK cells were transfected with the indicated plasmids, 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 similar groups based on ANOVA (P < 0.01) are indicated with one or two asterisks. The error bars indicate standard deviations. (D) 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 transfections were immunoblotted with antibodies against β-actin as a control for protein input levels in immunoprecipitation.