Stat2

DB and OCI-ly10 were isolated and lysed in lysis buffer (200 μl) (Sigma Aldrich, Shanghai, China). Protein lysates (10 μg) were electrophoresed on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes (BioRad, Hercules, CA, USA). Membranes were blocked for non-specific binding using 5% non-fat dried milk and left overnight at 4 °C rocking at low speed with IFNAR1 (ab45172, Abcam), IFNAR2 (ab190664, Abcam), p-STAT1 (9167, Cell Signaling Technology), STAT1 (14994 Cell Signaling Technology), p-STAT2 (4108, Cell Signaling Technology), STAT2 (72604, Cell Signaling Technology), p-STAT3 (9145, Cell Signaling Technology), STAT3 (9139, Cell Signaling Technology), OX40L (ab264466, Abcam), LC3A/B (4108, Cell Signaling Technology), p62 (88588, Cell Signaling Technology), and β-actin primary antibody (3700, Cell Signaling Technology). Horseradish peroxidase conjucated antibody was used as secondary detection. Immunocomplexes were visualized with an enhanced chemiluminescence detection kit.

Western blotting was performed using a standard protocol established in our laboratory. Infected Huh-7.5 cells were washed twice with PBS and then lysed in ice-cold RIPA buffer. Total protein content of the extract was quantified using a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA). Equal amounts of proteins were loaded on SDS-PAGE gels and Western blotting was carried out using antibodies to STAT1, p-STAT1, STAT2, p-STAT2, HSC70, β-actin and GAPDH (Cell Signaling Danvers, MA); as well as LAMP2A (Abcam).

STAT1 and STAT2 signaling was studied in A549 cells as previously described [61 (link)]. Briefly, A549 cells were infected with the indicated ZIKV strain at an MOI of 0.1 and 1 (based on Vero cell titration). At 48hpi, cells were pulse treated with 1000 IU/mL of recombinant human IFNβ (PBL Assay Science) for 30 minutes and whole-cell lysates were collected in modified radioimmunoprecipitation assay buffer supplemented with Halt Protease Inhibitor Cocktail (ThermoFisher) and phosphatase inhibitor cocktail II (Calbiochem). Western blot analysis was performed to detect STAT1 phosphotyrosine residue 701 (Cell Signaling), total STAT1 (Cell Signaling), STAT2 phosphotyrosine residue 689 (Upstate, EMD Milipore), total STAT2 (Cell Signaling), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Cell Signaling). Protein expression levels were quantified using Image Lab software. For analysis of antiviral effector proteins within human moDCs, 4e5 cells were used per condition and protein lysates were collected as described for A549 cells. The following antibodies were obtained from Cell Signaling: RIG-I, MDA5, LGP2, STAT1, STAT2, IFIT1, viperin, and GAPDH. The IFIT3 antibody was kindly provided by Dr. G. Sen.