Fig 2.

Mx1 inhibits the interaction between PB2 and NP. (A) HEK293T cells were transfected in triplicate with PB1, PA, and NP expression plasmids (25 ng each), together with pHW-NSLuc (100 ng) and pRL-CMV (25 ng). In addition, 25 ng pCAXL-PB2 or pCAXL-PB2V5 and 125 ng of pCAXL or pCAXL-Mx1 were cotransfected. The relative luciferase activity in the lysates was determined 48 h after transfection. Bars represent the average of the triplicates, and the error bars depict one standard deviation. This graph is representative of two independent experiments. (B) HEK293T cells were transfected with plasmids for expression of PB1, PB2V5, PA, and NP (1 μg each) and with pHW-NSLuc (1 μg). In addition, increasing amounts of pCAXL-Mx1 were cotransfected (0 μg, 0.5 μg, 1 μg, 2 μg, or 5 μg). Total lysates were made 24 h after transfection, and PB2V5 and NP were immunoprecipitated (IP) with anti-V5 and anti-NP antibodies, respectively. Proteins were visualized by Western blotting with antibodies recognizing the V5 tag, NP (anti-RNP antibody), and Mx1. (C) HEK293T cells were transfected in triplicate with plasmids for expression of PB1, PB2, PA, and NP (25 ng each), together with pHW-NSLuc (100 ng) and pRL-CMV (25 ng). Increasing amounts of pCAXL-Mx1 were cotransfected (0 ng, 12.5 ng, 25 ng, 50 ng, or 125 ng). The normalized luciferase activity in the lysates was determined 48 h after transfection. Bars represent the average of triplicates, and the error bars depict one standard deviation.