Horseradish peroxidase hrp

HEK-293 and PBMC cell lysates were prepared with RIPA buffer (10 mM Tris–HCl [pH 7.4], 150 mM NaCl, 1% NP-40, 1 mM EDTA, 0.1% SDS, 1 mM dithiothreitol); 20 μg protein aliquots from each sample were denatured and size-fractionated by electrophoresis through SDS-containing, 4–12% gradient polyacrylamide gels (Thermo Fisher Scientific), SDS-PAGE. The proteins were then transferred onto nitrocellulose membranes using Trans-Blot Turbo Transfer System (Biorad). Full-length membranes or membranes cropped based on molecular weight were incubated for 16 h with the appropriate primary antibodies recognizing target proteins NF90 (BD Transduction Laboratories) EPS15L1 (OriGene Technologies), RGS14, EBNA1BP2 (Novus Biologicals), PSMD11, DDX39B, RBM6, NFKBID, ZFR, YWHAH, PSMA3, IRF3, IRF9 (Abcam), ACTB (Santa Cruz Biotechnology). After incubation with appropriate secondary antibodies conjugated with horseradish peroxidase (HRP) (Kindle Biosciences), protein signals were detected by enhanced chemiluminescence (Euroclone). Images of uncropped membrane are present in Supplemental Fig. S5.