Horseradish peroxidase conjugated anti mouse or anti rabbit igg antibody

Samples were separated by SDS-polyacrylamide (10-16.5%) gel electrophoresis and electroblotted onto a membrane filter (Immobilon-P; MilliporeSigma, Burlington, MA, USA). Membranes were blocked with Blocking One (Nacalai Tesque) for 30 min, followed by incubation at 20–25°C with rabbit polyclonal antibodies against FLAG (MBL), V5 (MBL), Myc (MBL), HA (MBL), MAVS (D5A9E) (#24930, Cell Signaling Technology, Danvers, MA, USA), TRIM25 (#13773, Cell Signaling Technology) or a mouse monoclonal antibody against RIG-I (D-12) (sc-376845, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) for 1 h. Membranes were then incubated at 20–25°C for 30 min with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG antibodies (GE Healthcare, Chicago, IL, USA). Protein bands were visualized using enhanced chemiluminescence Western Lightning Ultra Substrate (PerkinElmer, Waltham, MA, USA) and FUSION-Solo S Imaging System (Vilber Lourmat Sté, Collégien, France).

Cellular extracts were prepared as previously described [6 (link)]. Protein concentrations in supernatants were determined using CD Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA). We performed Pounceau staining to confirm correct protein transfer and used it as loading control where indicated. Western blots were performed with 20 μg of total protein extract, using antibodies against Cleaved Caspase 3 (9664S), phospho-Chk1 (Ser345, 133D3) (Cell Signaling, Danvers, MA, USA), phospho-Rpa2 (Ser4/Ser8, A300-245A-M, Thermo Fisher Scientific), phospho-histone H2AX (Ser139, 07-164, Millipore, Billerica, MA, USA), β-actin (A5441) (Sigma), E2F1 (sc-251), Parp-1 (sc-53643), Fas (sc-1023), FasL (sc-6237), Mcm2 (sc-9839), Cyclin A2 (sc-596), Chk1 (sc-7898), p53 (sc-126), and Hsp 90α/β (sc-13119) (Santa Cruz Biotechnologies, Santa Cruz, CA, USA). Immunocomplexes were visualized with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG antibodies (Amersham, GE Healthcare Bio-Sciences, Pittsburgh, PA, USA), followed by chemiluminescence detection (ECL, Amersham, GE Healthcare Bio-Sciences, Pittsburgh, PA, USA) with a ChemiDoc camera (Bio-Rad Laboratories, Hercules, CA, USA). Densitometry-based quantification was performed using Fiji software. Relative optical density was calculated by dividing the densitometry of the protein of interest with the respective loading control.