Flow cytometry

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 temperature with the following fluorescently conjugated primary antibodies prepared in 0.5% PBSA: pSTAT1-PE (no. 8062, 1:50, CST) and ZIKV NS5 AF647 (1:2,000, conjugated in-house). ZIKV NS5 was conjugated to AF647 using an Alexa Fluor 647 antibody labeling kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Following staining, cells were washed with 1× PBS and data were acquired on a Canto II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Flow cytometry data were analyzed in FlowJo (BD Biosciences).