Figure 2  Validation of the LC-MS/MS results by Western blot analysis. (A) Quantitative real-time PCR (qPCR) analysis of the relative mRNA expression level of ANAPC7 and IFIT1 in IPEC-J2 cells upon PDCoV infection. IPEC-J2 cells were mock infected or infected with the PDCoV CHN-HN-1601 strain at an MOI of 0.1 TCID50/cell and collected at 24 hpi. Total RNA was extracted and reverse-transcribed into cDNA for the subsequent analysis via qPCR. Fold-change values were calculated based on the 2–ΔΔCt method, using β-actin as the housekeeping gene. Error bars indicate the standard error of three independent experiments (Student’s t test; *p < 0.05). (B) The relative ratio of ANAPC7 and IFIT1 mRNAs normalized to β-actin between PDCoV- and mock-infected cells was calculated based on the qPCR data. The iTRAQ ratio (PDCoV/Mock) obtained by MS analysis was simultaneously shown as a comparison. (C) Western blot (WB) analysis of the expression of ANAPC7 and IFIT1 proteins in IPEC-J2 cells upon PDCoV infection. IPEC-J2 cells were mock infected or infected with the PDCoV CHN-HN-1601 strain at an MOI of 0.1 TCID50/cell. At 24 hpi, the cells were harvested and processed for WB analysis using rabbit anti-ANAPC7, mouse anti-IFIT1 polyclonal antibodies and the mAb 1A3 specific for PDCoV. β-Actin was included as an internal loading control. The images shown are representatives of three independent experiments. (D) The optical intensity ratio between the corresponding bands (PDCoV-infected band/Mock band) was measured by densitometric scanning and normalized to the intensity of the β-actin bands in each experiment. The iTRAQ ratio (PDCoV/Mock) obtained by MS analysis was simultaneously shown as a comparison.