Anti-TGEV Miller Strain Infection Effect of Lactobacillus plantarum Supernatant Based on the JAK-STAT1 Signaling Pathway

Wang, Kai; Ran, Ling; Yan, Tao; Niu, Zheng; Kan, Zifei; Zhang, Yiling; Yang, Yang; Xie, Luyi; Huang, Shilei; Yu, Qiuhan; Wu, Di; Song, Zhenhui

Transmissible gastroenteritis virus (MOI = 0.1) was infected to IPEC-J2 cells that had been treated with Lp-1s for 1.5 h, and then cell samples at 12, 24, and 48 hpi were collected to produce protein lysates. In addition, TGEV (MOI = 0.1) was infected into IPEC-J2 cells cultured in RPMI 1640 for 1.5 h as the TGEV control group and the proteins were extracted at the same time points as the Lp-1s group. The protein concentration was determined using the bicinchoninic acid method and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. The separated proteins were transferred to a nitrocellulose membrane (Bio-Rad). The membranes were blocked using 5% skimmed milk and incubated with the following primary antibodies: Anti-STAT1 rabbit polyclonal antibodies (British Biorbyt Company), anti-phospho-(p)STAT1 (Tyr701) rabbit polyclonal antibodies (Biorbyt), anti-ZAP rabbit polyclonal antibodies (Abcam), anti-PKR rabbit polyclonal antibodies (Abcam), anti-(p)PKR (T446) rabbit polyclonal antibodies (Abcam), and anti-β-tubulin rabbit polyclonal antibodies (Proteintech). Secondary antibodies comprised goat anti-rabbit immunoglobulin (H + L). The immunoreactive protein bands were visualized using the Vilber fusion FX5 imaging system (VILBER), and the grayscale values of each band were analyzed by GraphPad Prism.