Subcellular fractionation and Western blot analysis

The Huh7 cells grown in 6-well plates at 80% or 90% were infected with mock or DENV-2 for 24 h. Then, the Huh7 cells were washed 3 times using cold 1X PBS, and the cells were fractionated following the Abcam Cell Fractionation Kit (ab109719) instructions. The cytoplasmic and nuclear fractions were stored at -80°C until use. The extracts from cytoplasmic fractions were quantified using the BCA Protein Assay (Thermo Scientific). SDS-PAGE separated thirty micrograms of protein, transferred to nitrocellulose membranes (Bio-Rad), and blocked with 10% nonfat milk in PBST (1X PBS/0.01% Triton X-100) for 1 h at RT. The immunoblotting was performed with the mouse anti-KPNA1 (1:1000; sc-517105), mouse anti-KPNA2 (1:1000; sc-136204), mouse anti-GAPDH (1:1000; sc-47724), mouse anti-lamina A/C (LMNA 1:1500; sc-376248) as nuclear fraction control or mouse anti-calreticulin (CRT) polyclonal antibody (1:1500; sc-6468) as cytoplasmic fraction control. HRP-conjugated goat anti-mouse or anti-rabbit IgG antibodies (1:5000; Cell Signaling) with 5% nonfat milk in PBST were used as secondary antibodies. The proteins were visualized using the Super Signal West Femto Chemiluminescent Substrate (Thermo Scientific), and the densitometric analysis was performed with the ImageJ software.