The interaction between VPg and eIF4E in RK13 cells based on the mammalian two-hybrid assay.

RK13 cells (2×105 cells per well in a 6-well plate) were co-transfected with the pACT (1 μg) and pBIND (1 μg) plasmids, pACT-VPg (1 μg) and pBIND (1 μg) plasmids, and pACT-VPg (1 μg) and pBIND-eIF4E (1 μg) plasmids, respectively. RK13 cells co-transfected with the pACT-MyoD (1 μg) and pBIND-ID (1 μg) plasmids served as positive controls [25]. A blank control was also used. In addition, all of the groups were transfected with pGL4.75 (0.2 μg). The cells were lysed at 48 h post-transfection, and Fluc activity was evaluated based on RLUs and normalized according to the results obtained for a co-transfected plasmid encoding the Renilla luciferase. The experiments were conducted in triplicate, and similar results were obtained from three independent experiments. The Student’s t test and ANOVA were applied for the statistical analyses, P <0.01 was considered as extremely significant different (**).