Anti zap

Proteins were resolved through SDS-PAGE using 4–15% precast Mini-PROTEAN TGX Gels (Bio-Rad) and NuPAGE MOPS SDS running buffer (Thermo Fisher Scientific) before transferring to a PVDF membrane (Bio-Rad). Immunodetection was achieved with 1:5,000 anti-ZAP (Abcam, Cambridge, United Kingdom); 1:5,000 anti-TRIM25 (BD Biosciences), 1:1,000 anti-HA (Roche Life Science), 1:5000 anti-V5 (Invitrogen), 1:2,500 anti-myc (Cell Signaling Technology, Danvers, MA), 1:20,000 anti-FLAG (Sigma-Aldrich), 1:500 anti-G3BP1 (Santa Cruz, Dallas, TX), 1:1,000 anti-G3BP2 (Assay Biotech, Fremont, CA), 1:1,000 anti-UPF1 (Cell Signaling Technology), 1:10,000 anti-NME1 (Abcam), 1:2,000 anti-PABPC4 (Proteintech, Rosemont, IL), and 1:20,000 anti-actin-HRP (Sigma-Aldrich). Primary antibodies were detected with 1:20,000 goat anti-mouse HRP (Jackson ImmunoResearch, West Grove, PA), 1:20,000 goat anti-rabbit HRP (Thermo Fisher Scientific), or 1:20,000 donkey anti-rat HRP (Jackson ImmunoResearch). Proteins were resolved on a 4–15% Mini-PROTEAN TGX gel (Bio-Rad, Hercules, CA) and visualized using ProSignal Pico ECL Reagent (Genesee Scientific, San Diego, CA) on a ChemiDoc (Bio-Rad). Quantification of western blots was performed using ImageJ.

Total protein was isolated by cell lysis using RIPA lysis buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris-HCl) supplemented with cOmplete Mini Protease Inhibitor Cocktail (Roche Life Science), followed by quantification with a colorimetric protein assay (Bio-Rad). Proteins were resolved by SDS-PAGE using Tris-glycine-SDS electrophoresis buffer (25 mM Tris, 192 mM glycine, and 0.1% SDS) and 4–15% precast Mini-PROTEAN TGX Gels (Bio-Rad) before transferring to nitrocellulose membranes (Bio-Rad). Immunodetection was performed with 1:5000 anti-ZAP (catalog number ab154680; Abcam, Cambridge, UK) and 1:20,000 anti-actin-HRP (catalog number A3854; Sigma-Aldrich, St. Louis, MO, USA). The ZAP primary antibody was detected with 1:20,000 goat anti-rabbit-HRP (Thermo Fisher Scientific). Proteins were visualized with ProSignal Pico ECL Reagent (Genesee Scientific) on a ChemiDoc imager (Bio-Rad).

48 h post-transfection, cells were lysed in radioimmunoprecipitation (RIPA) buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% sodium dodecyl sulfate (SDS), 1% Triton X-100, 1% sodium deoxycholate). The supernatant was filtered through a 0.45-μm filter. Virions were pelleted through a 20% sucrose cushion in PBS solution for 2 h at 20,000 × g. The pellet was resuspended in 2× loading buffer (60 mM Tris-HCl, pH 6.8, 10% β-mercaptoethanol, 10% glycerol, 2% SDS, 0.1% bromophenol blue). Cell lysates and virions were resolved by SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. Protein bands were detected using the LI-COR infrared imaging system (LI-COR UK).
The antibodies used in this study were 1:50 HIV-1 anti-p24^Gag (183-H12-5C),⁶⁹ (link) 1:1,000 anti-heat shock protein (HSP)90 (Santa Cruz Biotechnology; sc7947), 1:5,000 anti-ZAP (Abcam; ab154680), 1:1,000 anti-GFP (Sigma-Aldrich; 11814460001), 1:10,000 anti-MLV p30^Gag (ATCC; CRL-1912)⁷⁰ (link), 1:10,000 Dylight 800-conjugated anti-mouse/rabbit secondary antibodies (Cell Signaling Technology), and IRDye 800CW goat anti-rat immunoglobulin G (IgG; LI-COR Biosciences).