Ab154680

Samples were mixed with 2× SDS loading buffer containing 10% β-mercaptoethanol and heated to 95°C for 10 min. Samples were loaded onto a SDS-8% PAGE gels. Proteins were size separated by electrophoresis and semi-dry blotted onto Immobilin-P membranes (Merck Millipore). The membranes were stained for tubulin (1:2000 in 1% milk powder-PBS-Tween; A11126; Molecular Probes) and human ZAP (1:5000 in 1% milk powder-PBS-Tween; ab154680, Abcam). Finally, the membranes were stained with secondary alkaline phosphatase-conjugated antibodies (α-mouse, A5153, Sigma-Aldrich; α-rabbit, D0487, Dako) and developed with nitroblue tetrazolium (NBT)/BCIP (5-bromo-4-chloro-3-indolylphosphate) (Roche).

Virus-infected or plasmid-transfected cells were harvested and lysed with RIPA lysis buffer (25 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, and 5% glycerol, supplemented with a protease inhibitor cocktail (Halt™ Protease Inhibitor Cocktail, Thermo Fisher Scientific, MA, United States). The mixture was centrifuged at 12,000 g, 4°C for 10 min and the resulting supernatants were mixed with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer and boiled for 5 min. Proteins from the cell lysates were separated using SDS-PAGE, and then transferred to nitrocellulose membranes (Bio-Rad Laboratories, CA, United States). The membrane was blocked with 5% skim milk prior to incubation with the indicated primary antibodies. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Biolegend, CA, United States) or (HRP)-conjugated donkey anti-rabbit IgG (Biolegend, CA, United States) were used as secondary antibodies. Primary antibodies used in this study included mouse-anti-Myc (Thermo Fisher Scientific, MA, United States), rabbit-anti-FLAG (ab1162, abcam, Cambridge, United Kingdom), rabbit-anti-HA (ab9110, abcam, Cambridge, United Kingdom), and mouse-anti-PEDV-N (SD 6–29, Medgene labs, SD, United States), rabbit polyclonal anti-Zinc finger antiviral protein (ab154680, abcam, Cambridge, United kingdom).

Polypeptides were resolved by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to a nitrocellulose membrane (GE Healthcare). Immunodetection was achieved with 1:5000 anti-ZAP (ab154680; Abcam), 1:5000 anti-NZAP (mouse monoclonal 23D1.1; see below), 1:5000 anti-V5 (MA5-15253; Thermo Fisher Scientific), 1:5000 anti-TRIM25 (610570; BD Biosciences), 1:1000 anti-HA (clone 3F10; Roche), 1:500 anti-ubiquitin (P4D1; Santa Cruz Biotechnology), or 1:50,000 anti-actin-HRP (A3854; Sigma) antibodies. The primary antibodies were detected with 1:20,000 goat anti-mouse HRP (115-035-146; Jackson ImmunoResearch), 1:20,000 goat anti-rabbit HRP (31462; Thermo Fisher Scientific), or 1:20,000 donkey anti-rat HRP (712-035-153; Jackson ImmunoResearch). Mouse monoclonal antibodies to rat NZAP previously generated [51 (link)] were screened for cross-reactivity to human NZAP. The clone 23D1.1 was submitted for production and purification by Cell Essentials. Anti-GFP antibody (rabbit polyclonal) was generated previously [52 (link)]. The proteins were visualized by ECL Prime Western Blotting Detection Reagent (GE Healthcare) or SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific).