Prosignal pico ecl reagent

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).

Total proteins, prepared by cells direct lysis in SDS protein sample buffer, were separated in 4–12% Bis-TRIS NuPAGE gels (Thermo Fisher Scientific) in 1 × MES buffer and transferred onto a nitrocellulose membrane. The in-house produced anti-ORF57 polyclonal antibody was used to ORF57 expression [51 (link)]. The anti-ORF50 antibody was generously provided by Dr. Yoshihiro Izumiya (University of California, Davis). The anti-Cas9 mouse monoclonal antibody was purchased from Cell Signaling (cat. no. 14697). The anti-FLAG polyclonal antibody (cat. no. F7425, Sigma-Aldrich) was used for FLAG-ORF70 and anti-c-myc clone 9E10 antibody (a generous gift from Dr. Xuefeng Liu, Georgetown University) for K3-myc fusion detection in the reporter assay. Mouse monoclonal anti-K8 antibody were obtain from ProMab Biotechnologies (cat. no. 20015). In-house generated culture media from the mouse hybridoma clone (7B4) culture expressing anti-SRSF3 antibody was used for SRSF3 detection. The cellular β-tubulin detected by mouse monoclonal antibody (cat. no. T5201, Sigma-Aldrich) was used as a loading control. The secondary horseradish peroxidase-labeled secondary antibodies (Sigma-Aldrich) were used for signal development using ProSignal Pico ECL Reagent (GeneSee Scientific) captured by Chemidoc Touch Imager (BioRad).