P stat2

For protein analyses the cells from different blood donors were pooled to get sufficient amounts of proteins, and whole cell lysates were prepared in the passive lysis buffer of Dual Luciferase Assay Kit (Promega) containing 10 mM Na₃PO₄. Equal amounts of proteins (10–30 ug/lane) were separated on SDS-PAGE and transferred to Hybond-P polyvinylidene difluoride (PVDF) membranes (Amersham Biosciences). The membranes were blocked with 5% milk protein in PBS. Antibodies against IRF3 and MxA were as previously described^{21 (link),22 (link)} and antibodies against ZIKV NS5 were prepared as described above. The staining was done in blocking buffer at RT for 1 h. Antibodies against phosphorylated IRF3 (P-IRF3, #4947), STAT2 (#72604), P-STAT2 (#88410) and GAPDH (#2118) were from Cell Signaling Technology, for β-Actin (SC-10731) from SantaCruz Biotechnology, and the stainings were done in Tris-buffered saline, pH 7.4 containing 5% BSA at +4 °C overnight. HRP-conjugated antibodies (Dako) were used in the secondary staining at RT for 1 h. Protein bands were visualized on HyperMax films using an ECL plus system (GE Healthcare).

Total protein was isolated from cells or tissue samples using RIPA lysis buffer. The protein concentration was determined using the Bradford assay. Equal amounts of total protein were separated by SDS-PAGE electrophoresis, transferred to PVDF membranes, and blocked with 5% skim milk powder at room temperature for 1 h. Next, the PVDF membranes were washed with TBST containing NaCl, Tris-HCl, and Tween-20 and incubated with primary antibodies against target proteins at 4 °C overnight, followed by two washes with TBST. TMEM2 antibody was purchased from Aviva Systems Biology. Antibodies against p-Tyk2, Tyk2, p-JAK, JAK, p-STAT1, STAT1, p-STAT2, STAT2, IRF9, and GAPDH were obtained from Cell Signaling Technology. PVDF membranes were incubated with the appropriate secondary antibodies at room temperature for 1 h and washed three times with TBST. Protein bands were visualized by chemiluminescence (ECL, Forevergen, Guangzhou, China).

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.