Ecl plus system

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).

Log-phase cultures of cells grown in YEP + 2% glucose were harvested and treated with 5% trichloroacetic acid (TCA) at 4°C overnight. TCA treated cell pellets were washed with acetone, air dried, and resuspended in lysis buffer (10 mM Tris, 1 mM EDTA, 2.75 mM DTT, pH = 8). Cells were lysed by bead-beating using a Multivortexer (max speed, 20 min) and glass beads at 4°C and followed by boiling in SDS PAGE protein loading buffer for 5 min. Lysates were clarified by centrifugation and were resolved on a 15-well NuPAGE 4–12% Bis-Tris protein gel (Thermo Fisher Scientific) prior to transfer onto nitrocellulose membranes. GFP-Mob1 and variants were detected using an anti-GFP antibody (Clontech, JL-8) at a 1:1000 dilution. Nud1-13myc and Cfi1/Net1-3myc were detected using an anti-Myc antibody (Abcam, 9E10) at a 1:500 dilution. Mob1-V5-TurboID was detected using an anti-V5 antibody (Invitrogen) at a 1:2000 dilution. Kar2 was detected using a rabbit anti-Kar2 antiserum at a 1:200,000 dilution. Secondary antibodies were used at a 1: 10,000 dilution. Blots were imaged using the ECL Plus system (GE Healthcare).

Cells were washed three times with phosphate-buffered saline (PBS), scraped from the culture dishes, and solubilized in RIPA buffer (Thermo Scientific, Lafayette, CO, USA) with 10 μM protein inhibitors (Thermo). The samples were then centrifuged at 20,000 g for 15 min, and the supernatants were collected. Protein determination was performed using the BCA assay kit (Pierce, Rockford, IL, USA). Samples were loaded on 4–20% gradient polyacrylamide gel (Bio-Rad) and transferred to PVDF membranes. After blocking in 10 mM Tris-HCl pH 7.4 and 150 mM NaCl (TBS) containing 5% non-fat milk, membranes were incubated with primary antibodies overnight at 4 °C. Primary antibodies for GH (1:500, R&D), PLZF (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA), Atp6l (1:500, Abcam, Cambridge, MA, USA) and β-actin (1:1000, Santa Cruz Biotechnology) were used for the study. Polyclonal rabbit anti-human PRR (1:1000, ProteinExpress, Chiba, Japan), which recognizes extracellular domains, has been previously described8 (link). After extensive washing, the membrane was incubated with peroxidase secondary antibody for 1 h at room temperature, and the complexes were detected using an enhanced chemiluminescence (ECLplus) system (GE Healthcare, Tokyo, Japan). The signals of each blot were visualized and quantitatively analyzed by ImageQuant LAS 4000 (GE Healthcare).