2.10  Western Blot Analysis
To further validate the identified DEPs, Western blot was performed to detect ANAPC7 and IFIT1 protein expression levels using β-actin as an internal control. Briefly, total cellular proteins were extracted from both mock- and PDCoV-infected IPEC-J2 cells at 24 hpi. The resulting protein samples were separated on 12% SDS-PAGE gels and transferred onto 0.22 μm poly(vinylidene fluoride) (PVDF) membranes (Millipore, Bedford, MA, USA) using a semidry electrophoretic transfer cell (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 5% (w/v) nonfat dry milk overnight at 4 °C and then probed with either anti-ANAPC7 (1:1000), anti-IFIT1 (1:1000), or anti-β-actin (1:5000) primary antibodies at 37 °C for 1 h. After thorough washing with PBST, the membranes were incubated with the corresponding HRP-conjugated secondary antibodies (1:8000) for 1 h at 37 °C. The target protein blots on the membranes were developed with an enhanced chemiluminescence detection kit (Thermo Fisher Scientific), and images were taken using a ProteinSimple FluorChem E image system (Santa Clara, CA, USA).