Assays

For immunoblot analysis, protein lysates were incubated with 4× Laemmli buffer (Bio-Rad) containing 10% β-mercaptoethanol (Thermo Fisher-Life Technologies) at 95°C for 3 min. Approximately 7 to 10 μg of total protein was loaded per lane onto 4 to 20% Criterion TGX gradient gels (Bio-Rad) and electrophoresed at ~96 V in 1× sodium dodecyl sulfate (SDS) running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS). Proteins were then transferred onto nitrocellulose membranes (Fisher Scientific) at 90 V for ...

A549 cells were seeded on no. 1 glass coverslips in a 24-well plate at a density of 1 × 105 cells per well. The next day, cells were mock infected or infected with ZIKV strains and fixed with 4% formaldehyde (Fisher Scientific) in filtered 1× TBS for 30 min at room temperature. Cells were then washed with filtered 1× TBS and permeabilized and blocked in 5% NGS in 1× TBS (5% NGS/TBS) containing 0.1% Triton X-100 for 30 min at 4°C. For pSTAT1 staining, cells were permeabilized with 100% ice-cold ...

Cells were seeded, infected, or transfected and treated with IFN-β as described above. Following IFN-β treatment, A549 RIG-I KO and HEK293T cells were pelleted and fixed with 4% formaldehyde in 1× PBS at room temperature for 15 min. Cells were then washed with excess 1× PBS, pelleted, and resuspended in 1× PBS for storage at 4°C. Fixed cells were permeabilized with 90% ice-cold methanol in 1× PBS for at least 10 min on ice. Cells were washed with excess 1× PBS and incubated for 1 h at room ...