Donkey anti rat hrp

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.

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).