Multi gauge version 3

The cell membranes were disrupted with cell lysis buffer (10 mM Tris-HCl, pH 7.8, 1 mM ethylenediaminetetraacetic acid (EDTA), 1% NP-40, 0.15 M NaCl), including cOmplete Mini (Roche Diagnostics, Tokyo, Japan) at 20 h after infection. The cell lysates were resolved by electrophoresis on 12.5% SuperSep gels (Fujifilm WAKO Pure Chemical Corporation) and Western blotting onto Immobilon-P membranes (Millipore, Tokyo, Japan). Non-specific protein binding was blocked with 5% non-fat dry milk for 1 h, and then the membranes were incubated with anti-feline coronavirus nucleocapsid (N) antibody (FIPV3-70; MyBioSource, San Diego, CA, USA) or anti-glyceraldehyde-3-phosphate dehydrogenase antibody (GAPDH, 6c5 clone; Calbiochem, Tokyo, Japan) for 1 h. Antigen signals were visualized by reacting proteins on the membranes with horseradish peroxidase-conjugated anti-mouse IgG antibody (Promega Corporation, Madison, WI, USA), followed by an enhanced chemiluminescence substrate (ImmunoStar LD; Fujifilm WAKO Pure Chemical Corporation, Tokyo, Japan), according to the manufacturer’s protocol. Signals were detected using the ImageQuant LAS 4000-mini Imaging System (GE Healthcare Life Sciences, Marlborough, MA, USA) and analyzed using Multi Gauge Version 3.0 software (GE Healthcare Life Sciences, MA, USA).

Cell membranes were disrupted using cell-lysis buffer [10 mM Tris/HCl, pH 7.8, 1 mM EDTA, 1 % (w/v) NP-40, 0.15 M NaCl] containing the Complete Mini reagent (Roche Diagnostics). Lysate proteins were resolved by subjecting them to electrophoresis through 12.5 % SuperSep gel (Wako Laboratory Chemicals). The proteins were electrophoretically transferred from the gels to Immobilon-P membranes (Millipore) that were previously treated with 5 % (w/v) skimmed dry milk for 1 h at room temperature to minimize nonspecific binding. Membranes were then incubated for 1 h with the primary antibodies. Immune complexes were visualized using an anti-mouse IgG (Promega) or anti-rabbit IgG antibodies (Promega), each of which was conjugated to HRP, followed by incubation with an enhanced chemiluminescence substrate (SuperSignal West Femto Maximum Sensitivity Substrate; Thermo Scientific), according to the manufacturer’s protocol. Signals were detected using the ImageQuant LAS 4000-mini Imaging System (GE Healthcare Life Sciences) and analysed by Multi Gauge Version 3.0 software (GE Healthcare Life Sciences).