Immunoblot analysis

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 1 h at 4°C in 1× Towbin transfer buffer (25 mM Tris, 192 mM glycine, 0.01% SDS, 20% methanol). Membranes were blocked for 1 h at room temperature in Tris-buffered saline (TBS)-based Intercept blocking buffer (LI-COR, Lincoln, NE, USA) and stained overnight at 4°C with the following primary antibodies diluted in blocking buffer: pIRF3 S386 (no. ab76493; 1:1,000; Abcam, Cambridge, UK), pIRF3 S396 (no. 4947; 1:1,000; Cell Signaling Technology [CST], Danvers, MA, USA), IRF3 (no. 4302; 1:1,000; CST), IκBα (no. 4814; 1:1,000; CST), IFIT1 (no. 971; 1:1,000; in-house), ZIKV NS5 (no. GTX133327; 1:2,500; GeneTex, Irvine, CA, USA), ZIKV NS1 (no. ARG65781, 1:1,000, Arigo Biolaboratories, Hsinchu City, Taiwan), ZIKV capsid (no. GTX133317, 1:1,000, GeneTex), pSTAT1 (no. 7649; 1:1,000; CST), STAT1 (no. 9172; 1:1,000; CST), STAT2 (no. 72604; 1:1,000; CST), IFITM1 (no. 60074-1-1g; 1:1,000; ProteinTech Group, Rosemont, IL, USA), OAS1 (no. 14498; 1:1,000; CST), Mx1 (no. 340B; 1:500; in-house), RIG-I (no. AG-20B-0009-C100; 1:1,000; Adipogen, San Diego, CA, USA), and actin (no. MAB 1501; 1:1,000; Sigma-Aldrich). The next day, membranes were washed with TBS containing Tween 20 (TBST), and Alexa Fluor 680-conjugated (715-625-151 or 711-625-152) or Alexa Fluor 790-conjugated secondary antibodies (711-655-152 or 715-655-150) (all from Jackson ImmunoResearch) diluted 1:10,000 in blocking buffer were added and incubated for 1 h at room temperature. Prior to imaging, membranes were washed with TBST followed by 1× TBS. Membranes were imaged on an Odyssey CLx imager (LI-COR). If necessary, membranes were stripped with 5× NewBlot IR stripping buffer (LI-COR) diluted 1:5 in dH2O and reprobed. Protein abundance was quantified in Fiji and normalized to actin abundance.