FIG 7.

Silencing RRP1B reduces RdRp binding to mRNA in vivo. HEK293T knockdown cells were cotransfected with plasmids expressing HA-tagged PB1, HA-tagged PB2, HA-tagged PA, and pFlu-NP, which produces NP protein and NP vRNA. At 24 h posttransfection, the cells were harvested for RIP assays as described in Materials and Methods. The cell lysates were subjected to immunoprecipitation using anti-HA agarose to isolate the RdRp-mRNA complex. (A) The cell lysates and immunoprecipitated proteins were resolved on SDS-PAGE and immunoblotted with anti-HA antibody. (B, C, and D) The levels of input (left) and coprecipitated (right) cellular mRNAs of GAPDH (B), β-actin (C), and U2 snRNA (D) were determined by qRT-PCR. The values represent the means ± SD of the results of three independent experiments. *, P < 0.05, and **, P < 0.01 compared with controls.