Anti pin1 antibody

The cell membranes were disrupted with cell lysis buffer [10 mM Tris–HCl, pH 7.8, 1 mM ethylenediamine tetraacetic acid (EDTA), 1% NP-40, and 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 (WAKO, Tokyo, Japan) and Western blotted on to Immobilon-P membranes (Millipore, Tokyo, Japan). Non-specific protein binding was blocked with 5% non-fat dry milk, and then the membranes were incubated with the primary antibodies [anti-FCoV nucleocapsid (N) antibody (FIPV3-70; MyBioSource, CA, USA), anti-c-Myc antibody (Santa Cruz Biotechnology, CA, USA), anti-Pin1 antibody (Cell Signaling Technology, Tokyo, Japan), anti-Cyp B (Thermo Fisher Scientific, Yokohama, Japan), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Calbiochem, CA, USA)] for 1 h. Antigen signals were visualized by reacting proteins on the membranes with horseradish peroxidase-conjugated anti-mouse IgG antibody (Promega) and/or anti-rabbit IgG antibody (Promega) followed by an enhanced chemiluminescence substrate (SuperSignal West Femto Maximum Sensitivity Substrate; Thermo Fisher Scientific) according to the manufacturer's protocol.