*5.7. Quantitative Real-Time PCR*

As per the manufacturer's protocol, total RNA was isolated from cells via a Trizol reagent. Nanodrop lite (Thermo Inc, Waltham, MA, USA) was used to examine the concentrations of RNA. The reverse transcription was accomplished via Super-Script III First-strand cDNA Synthesis Mix (Thermo Inc, USA). Real-time PCR was performed with SybrGreen qPCR Mastermix (Thermo Inc, USA). The overall samples were assayed three times. The reaction mixtures were incubated in a 7900 fast real-time PCR system (Applied Biosystems, Foster City, CA, USA)). The program comprised of 1 cycle at 95 ◦C for 120 s, 40 cycles at 94 ◦C for 20 s, 60 ◦C for 20 s, and 72 ◦C for 30 s. The gene relative expression levels were calculated according to the 2−ΔΔCT method. In real-time PCR analysis, β-actin was used as a housekeeping gene to estimate levels of mRNA for normalization. The primer sequences were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China) and described in Table 1.




**Table 1.** *Cont*.

### *5.8. Immunofluorescence*

Phosphoryl-NF-κB p65 localization was quantified via an immunofluorescence technique. Paraformaldehyde (*v*/*v*, 1/25) was used for IPEC-J2 cells fixation for 30 min at a temperature of 37 ◦C. After a PBS wash (0.1 mM, pH7.4), permeabilised in 0.5% Triton (Triton×100, Sigma, Harz Lower Saxony, Germany) for 20 min, and for 20 min blocked with 5% BSA, as well as hatched with the anti-phosphoryl-NF-κB p65 antibody (diluted 1:100) for the whole night at a temperature of 4 ◦C. After washing with PBS (0.1 mM, pH7.4) for the second time, the secondary antibody was used to incubate the cells for 1 h at room temperature. Coverslips were washed two times via PBS (0.1 mM, pH7.4), and hatched with the goat anti-rabbit IgG antibody for 1 h in the absence of light, and hatched in a DAPI staining solution for 10 min. After that it was washed again in PBS. The fluorescence was monitored using an Olympus-fluoview ver.3.1 viewer (Olympus Corporation, Miyazaki Prefecture, Kyushu, Japan) [38].

### *5.9. Electrophoretic Mobility Shift Assays (EMSAs)*

NF-κB DNA-binding activity was examined by EMSA. The cytoplasmic and nuclear protein extraction kit (Jiangsu KeyGEN BioTECH Corp., Ltd., Nanjing, China) was used to prepare nuclear extract. The consensus nucleotide sequence for NF-κB was 5'-AGT TGA GGG GAC TTT CCC AGG C-3'. The EMSA binding reaction was performed by the EMSA kit (Jiangsu KeyGEN BioTECH Corp., Ltd.). A nuclear extract was incubated in a 5× binding reaction buffer containing the biotinylated probe. After incubated at room temperature for 20 min, the reaction mixture was electrophoresed on a non-denaturing 6.5% polyacrylamide gel and then transferred to a nylon membrane. The shifted mixture and the membrane were UV-cross-linked and the ECL kit used to detect was obtained from (Jiangsu KeyGEN BioTECH Corp., Ltd.). For the super shift assay, 1 μg of antibody against NF-κB p65 was added together with the nuclear extract.

### *5.10. Statistics Analysis*

For the calculation of protein expression of average optical density (OD), the Image-Proplus 6.0 Analysis Software (Media Cybernetics, Shanghai, China) were used. The obtained data were presented as means ± SD (n = 10). Statistical analysis was performed by the Statistical Program for Social Sciences (SPSS) software version 19.0 (IBM Corporation, Armonk, NY, USA). Analysis of variance (ANOVA) was performed for the multiple comparison of different groups. The histogram was designed by using the software of Graph Pad Prism version 5.0 (San Diego, California, CA, USA).

**Author Contributions:** Conceptualization, X.W.; Software, Y.H. and S.U.R.; Formal analysis, L.C.; Investigation, Y.L. and J.W.; Resources, X.W.; Data curation, Y.Z.; Writing—original draft, Y.Z.; Writing—review and editing, J.Z.; Project administration, X.W.; Funding acquisition, L.Z., X.C., and S.F.

**Funding:** The present study was supported by the National Natural Science Foundation of China (grant No. 31472250) and the Project of Modern Agricultural Industry and Technology System of Anhui Province (grant No. AHCYJSTX-05-07). We wish to thank anonymous reviewers for their kind advice.

**Conflicts of Interest:** The authors declare that they have no conflict of interest.
