Fig. 3

BinCARD1 increases the vRNP complex activity of IAV. A SiRNA knockdown of BinCARD in HEK293T cells. HEK293T cells were transfected with BinCARD siRNA1 or scrambled siRNA, and at 36 h p.t., the cells were subjected to RT‒qPCR analysis to determine the effect of BinCARD knockdown. ***P < 0.001. B The vRNP complex activity of IAV in BinCARD siRNA1-treated HEK293T cells. HEK293T cells treated with BinCARD siRNA1 or scrambled siRNA as described in A were further transfected with the set of plasmids for the evaluation of vRNP complex activity, including the four RNP protein expression constructs (PB2, PB1, PA, and NP) derived from WSN (H1N1) virus, pHH21-SC09NS F-Luc, and pRL-TK. Thirty-six hours later, a dual-luciferase assay was performed in which the relative firefly luciferase activity was normalized to the luciferase activity of the Renilla, the internal control. The expression of vRNP complex proteins was measured by western blotting with a mouse anti-PB2, PB1, PA, or NP mAb. ***P < 0.001. C The vRNP complex activity of IAV in HEK293T cells was increased with progressively increased BinCARD1 expression. HEK293T cells were transfected with the set of plasmids for the evaluation of vRNP complex activity as described in B, together with an empty vector or gradually increasing amounts of BinCARD1-expressing constructs. Thirty-six hours later, a dual-luciferase assay was performed as described in B. The expression of the BinCARD1 and vRNP complex proteins was measured by western blotting with a mouse anti-BinCARD1, PB2, PB1, PA, or NP mAb. **P < 0.01; ***P < 0.001. The data represent the mean ± SD of three independent experiments (A–C)