3.4  Validation of the DEPs by qPCR and Western Blot Analyses
To validate the obtained LC-MS/MS data, qPCR was performed to evaluate the transcription levels of two randomly selected DEPs, the downregulated ANAPC7 and the upregulated IFIT1. To this end, IPEC-J2 cells were mock-infected or infected with PDCoV at an MOI of 0.1. At 24 hpi, total cellular RNA was extracted from the cells and subjected to qPCR assays. As shown in Figure 2A, the level of mRNA encoding ANAPC7 and IFIT1 proteins was significantly downregulated and upregulated in PDCoV-infected cells, respectively, as compared to the mock-infected cells (p < 0.05). The qPCR results were in agreement with the MS data which were acquired by the iTRAQ approach (Figure 2B). For further confirmation of the proteomic data, the expression level of ANAPC7 and IFIT1 proteins in IPEC-J2 cells, which were infected exactly as the aforementioned conditions, was also tested by Western blot analysis. To track the progression of PDCoV infection, the mAb 1A3 that specifically recognizes PDCoV was utilized. As shown in Figure 2C, compared with the mock-infected IPEC-J2 cells, PDCoV significantly decreased the expression of ANAPC7 protein and its relative ratio to β-actin in the cells, whereas the expression of IFIT1 protein and its relative ratio to β-actin in the cells were significantly increased as a consequence of PDCoV infection. The original images of the entire PVDF membranes containing the target Western blots were included in Supplementary Figure S2. The Western blot results were also consistent with the MS data (Figure 2D). Taken together, these experimental results reveal that our quantitative proteomics data are quite reliable.
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.